Il-31 monoclonal antibody formulations

ABSTRACT

The invention provides humanized mouse anti-human IL-31 antibodies and antibody fragments that are capable of binding IL-31 and thereby neutralizing, inhibiting, limiting, or reducing the proinflammatory or pro-pruritic effects of IL-31.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/645,769, filed Mar. 12, 2015, which is a divisional of U.S. patentapplication Ser. No. 14/174,870, filed Feb. 7, 2014, now U.S. Pat. No.9,005,921, which is a continuation of U.S. patent application Ser. No.13/908,436, filed Jun. 3, 2013, now U.S. Pat. No. 8,685,669, which is adivisional of U.S. patent application Ser. No. 12/329,820, filed Dec. 8,2008, now U.S. Pat. No. 8,470,979, which claims the benefit of U.S.Provisional Application Ser. No. 61/012,362, filed Dec. 7, 2007, whichare all herein incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

IL-31, a newly identified cytokine, has been found to result indermatitis-like symptoms when over-expressed in mice. See, Dillon, etal., Nature Immunol. 5:752-760, 2004. These symptoms can be alleviatedby use of antagonists that block, inhibit, reduce, antagonize orneutralize the activity of IL-31, and include anti-IL-31 antibodies.Also see, U.S. patent application Ser. No. 10/352,554, filed Jan. 21,2003 (U.S Patent Publication No. 2003-0224487), now U.S. Pat. Nos.7,064,186, 7,425,325, and 7,459,293.

Monoclonal antibody technology has provided a vast array of therapeuticsas well as diagnostics for use in identifying and treating disease. Anumber of recombinant or biosynthetic molecules comprising rodentantigen-binding sites have been described. Particularly, moleculeshaving rodent antigen-binding sites built directly onto human antibodiesby grafting only the rodent binding site, rather than the entirevariable domain, into human immunoglobulin heavy and light chain domainshave been described. See, e.g., Riechmann et al. (1988) Nature332:323-327 and Verhoeyen et al. (1988) Science 239:1534-1536. Moleculeshaving an antigen-binding site wherein at least one of thecomplementarity determining regions (CDRS) of the variable domain isderived from a murine monoclonal antibody and the remainingimmunoglobulin-derived parts of the molecule are derived from humanimmunoglobulin have been described in U.K Patent Publication No. GB2,276,169, published Sep. 21, 1994. A number of single chainantigen-binding site polypeptides and single chain Fv (sFv) moleculeshave also been described. See, e.g., U.S. Pat. Nos. 5,132,405 and5,091,513 to Huston et al.; and U.S. Pat. No. 4,946,778 to Ladner et al.

Mouse anti-human IL-31 monoclonal antibodies have been describedpreviously in U.S. patent application Ser. No. 11/430,066, filed May 8,2006 (U.S. Patent Publication No. 2006-02752960) that describes mousemonoclonal antibodies that recognize human IL-31 and can be used togenerate chimeric antibodies. However, chimeric antibodies may causeimmunogenicity and humanized mouse-anti-human IL-31 antibodies aredesirable. Humanized antibodies generally have at least three potentialadvantages over mouse or in some cases chimeric antibodies for use inhuman therapy: (1) Because the effector portion is human, it mayinteract better with the other parts of the human immune system (e.g.,destroy the target cells more efficiently by complement-dependentcytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC));(2) The human immune system should not recognize the framework orconstant region of the humanized antibody as foreign, and therefore theantibody response against such an injected antibody should be less thanagainst a totally foreign mouse antibody or a partially foreign chimericantibody; and (3) Injected mouse antibodies have been reported to have ahalf-life in the human circulation much shorter than the half-life ofnormal antibodies (D. Shaw et al., J. Immunol., 138, 4534-4538 (1987)).Injected humanized antibodies will presumably have a half-life moresimilar to naturally occurring human antibodies, allowing smaller andless frequent doses to be given.

Thus, there is a need for molecules which provide humanized variableregion amino acid sequences for the mouse anti-human IL-31 antibodiesfor treating IL-31 mediated inflammation.

SUMMARY OF THE INVENTION

In an aspect of the present invention pertains to an isolated antibodythat binds to human IL-31, comprising: a) a humanized heavy chainvariable domain comprising CDRs consisting of amino acid sequences SEQID NO: 1, 2 and 3 respectively or consisting of amino acid sequences SEQID NO: 1, 4 and 3 respectively; b) a humanized light chain variabledomain comprising CDRs consisting of amino acid sequences of SEQ ID NO:5, 6 and 7. In another aspect, the invention pertains to antibodiesdescribed herein wherein: a) said humanized heavy chain variable domaincomprises framework regions FR1, FR2, FR3 and FR4 having an amino acidsequence at least 90% identical to the amino acid sequence selected fromthe group consisting of: 1) the amino acid sequence of SEQ ID NO: 8(FR1), 9 (FR2), 10 (FR3) and 11 (FR4) respectively; 2) the amino acidsequence of SEQ ID NO: 12 (FR1), 13 (FR2), 14 (FR3) and 15 (FR4)respectively; 3) the amino acid sequence of SEQ ID NO: 12 (FR1), 13(FR2), 16 (FR3) and 15 (FR4) respectively; and b) said humanized lightchain variable domain comprises framework regions FR5, FR6, FR7, and FR8having an amino acid sequence at least 90% identical to the amino acidsequence the framework regions are selected from the group consistingof: 1) the amino acid sequence of SEQ ID NO: 17 (FR5), 18 (FR6), 19(FR7) and 20 (FR8) respectively; and 2) the amino acid sequence of SEQID NO: 17 (FR5), 18 (FR6), 21 (FR7) and 20 (FR8) respectively. In anembodiment, said identity is at least 95%, even more preferably at least98%, most preferably at least 99%.

In another aspect the invention pertains to an isolated antibody asdescribed herein wherein amino acid at position 29 in FR1 of the heavychain is leucine and amino acid at position 32 in FR3 of the heavy chainis phenylalanine. In another aspect the invention pertains to anisolated antibody as described herein wherein amino acid at position 8in FR3 of the heavy chain is lysine and amino acid at position 15 in FR7of the light chain is tyrosine.

In a further aspect the present invention pertains to an isolatedantibody that binds to human IL-31, comprising: a) a humanized heavychain variable domain comprising CDR1 and CDR3 having amino acidsequence of SEQ ID NO: 1 and 3 respectively and CDR2 having amino acidsequence of AIYPGDGDTRYSXaa1Xaa2FXaa3G (SEQ ID NO: 22) wherein Xaa1 isglutamine or proline, Xaa2 is serine or lysine and Xaa3 is glutamine orlysine; said humanized heavy chain variable domain comprising frameworkregions FR1, FR2, FR3 and FR4 having an amino acid sequence at least 90%identical to the amino acid sequence of: SEQ ID NO: 8, 9, 10 and 11respectively, or SEQ ID NO: 12, 13, 14 and 15 respectively, or SEQ IDNO: 12, 13, 16 and 15 respectively; and with the proviso that amino acidat position 29 in FR1 is leucine and amino acid at position 32 in FR3 isphenylalanine; b) a humanized light chain variable domain comprisingCDRs consisting of amino acid sequences of SEQ ID NO: 5, 6 and 7 saidhumanized light chain variable domain comprising framework regions FR5,FR6, FR7 and FR8 having an amino acid sequence at least 90% identical tothe amino acid sequence of SEQ ID NO: 17, 18, 19 and 20 respectively, orat least 90% identical to the amino acid sequence of SEQ ID NO: 17, 18,21 and 20 respectively. In a aspect of the present invention, saididentity is at least 95%, even more preferably at least 98%, mostpreferably at least 99%. In another aspect the invention pertains to anisolated antibody as described herein wherein amino acid at position 8in FR3 of the heavy chain is lysine and amino acid at position 15 in FR7of the light chain is tyrosine. In another aspect the invention pertainsto an isolated antibody as described herein wherein: amino acid Xaa1 isglutamine, Xaa2 is lysine and Xaa3 is lysine; amino acid Xaa1 isproline, Xaa2 is serine and Xaa3 is glutamine; or amino acid Xaa1 isglutamine, Xaa2 is lysine and Xaa3 is glutamine.

In another aspect the invention pertains to an isolated antibody asdescribed herein wherein CDRs of the heavy chain variable domain consistof SEQ ID NO: 1, 4 and 3 respectively, CDRs of the light chain variabledomain consist of SEQ ID NO: 5, 6 and 7 respectively, framework regionsof the heavy chain variable domain consist of SEQ ID NO: 8, 9, 10 and 11respectively, and framework regions of the light chain variable domainconsist of SEQ ID NO: 17, 18, 19 and 20 respectively.

In another aspect the invention pertains to an isolated antibody asdescribed herein wherein CDRs of the heavy chain variable domain consistof SEQ ID NO: 1, 2 and 3 respectively, CDRs of the light chain variabledomain consist of SEQ ID NO: 5, 6 and 7 respectively, framework regionsof the heavy chain variable domain consist of SEQ ID NO: 12, 13, 14 and15 respectively, and framework regions of the light chain variabledomain consist of SEQ ID NO: 17, 18, 19 and 20 respectively.

In another aspect the invention pertains to an isolated antibody asdescribed herein wherein CDRs of the heavy chain variable domain consistof SEQ ID NO: 1, 2 and 3 respectively, CDRs of the light chain variabledomain consist of SEQ ID NO: 5, 6 and 7 respectively, framework regionsof the heavy chain variable domain consist of SEQ ID NO: 12, 13, 16 and15 respectively, and framework regions of the light chain variabledomain consist of SEQ ID NO: 17, 18, 21 and 20 respectively.

As it will be shown from the Examples and teachings herein, the presenceof both a leucine at position 29 in FR1 and a phenylalanine at position32 in FR3 of the humanized antibodies described herein, has a positiveimpact in terms of affinity and potency of said antibodies. As shown forexample in a Biacore assay, the binding affinity of humanized antibodiesbearing both a leucine at position 29 in FR1 and a phenylalanine atposition 32 in FR3 is better when compared to humanized antibodies whichbear another amino acid at these positions (compare for example clonesnumber 7, 10, 13 and 14 in Table 2 of Example 3). The positive impact ofthese amino acids at position 29 of FR1 and 94 of FR3 of the heavychain, both in terms of affinity and potency, has also been confirmed bytwo other biological tests (see Examples 4 and 5).

In an embodiment of the present invention, the antibody disclosed hereincomprises a heavy chain immunoglobulin constant domain selected from thegroup consisting of the constant region of an α, γ, μ, δ or ε humanimmunoglobulin heavy chain. In a embodiment of the present invention,the antibody disclosed herein comprises a heavy chain immunoglobulinconstant domain selected from the group consisting of a human IgG1constant domain; a human IgG2 constant domain; a human IgG3 constantdomain; a human IgG4 constant domain; a human IgM constant domain; ahuman IgE constant domain and a human IgA constant domain. In anembodiment, the human IgG4 constant domain is a mutated form stable insolution and with little or no complement activating activity. In anembodiment, the heavy chain immunoglobulin constant region domain is ahuman IgG4 constant domain with a Ser to Pro mutation at position 241(Kabat numbering).

In an embodiment, the immunoglobulin light chain constant region domainis selected from the group consisting of the constant region of a κ or λhuman immunoglobulin light chain. Preferably, the immunoglobulin lightchain constant region domain is the constant region of a κ humanimmunoglobulin light chain.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a shows an alignment of heavy chain anti-human IL-31 antibodysequences using the human VH1-46 germline gene as acceptor.

FIG. 1b shows an alignment of heavy chain anti-human IL-31 antibodysequences using the human VH5-51 germline as acceptor.

FIG. 1c shows an alignment of light chain anti-human IL-31 antibodysequences using the human VK1-A20 germline as acceptor. The amino acidsequences of the clones shown in FIGS. 1a-1c are listed in Table 1.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention in detail, it may be helpful to theunderstanding thereof to define the following terms:

As used herein, the term “antibodies” includes polyclonal antibodies,affinity-purified polyclonal antibodies, monoclonal antibodies, andantigen-binding fragments, such as F(ab′)₂, Fab proteolytic fragments,and single chain variable region fragments (scFvs). Geneticallyengineered intact antibodies or fragments, such as chimeric antibodies,Fv fragments, single chain antibodies and the like, as well as syntheticantigen-binding peptides and polypeptides, are also included. Non-humanantibodies may be humanized by grafting non-human CDRs onto humanframework and constant regions, or by incorporating the entire non-humanvariable domains (optionally “cloaking” them with a human-like surfaceby replacement of exposed residues, wherein the result is a “veneered”antibody). In some instances, humanized antibodies may retain non-humanresidues within the human variable region framework domains to enhanceproper binding characteristics. Through humanizing antibodies,biological half-life may be increased, and the potential for adverseimmune reactions upon administration to humans is reduced.

The term “chimeric antibody” or “chimeric antibodies” refers toantibodies whose light and heavy chain genes have been constructed,typically by genetic engineering, from immunoglobulin variable andconstant region genes belonging to different species. For example, thevariable segments of the genes from a mouse monoclonal antibody may bejoined to human constant segments, such as gamma 1 and gamma 3. Atypical therapeutic chimeric antibody is thus a hybrid protein composedof the variable or antigen-binding domain from a mouse antibody and theconstant domain from a human antibody, although other mammalian speciesmay be used.

As used herein, the term “immunoglobulin” refers to a protein consistingof one or more polypeptides substantially encoded by immunoglobulingenes. One form of immunoglobulin constitutes the basic structural unitof an antibody. This form is a tetramer and consists of two identicalpairs of immunoglobulin chains, each pair having one light and one heavychain. In each pair, the light and heavy chain variable regions aretogether responsible for binding to an antigen, and the constant regionsare responsible for the antibody effector functions.

Full-length immunoglobulin “light chains” (about 25 Kd or 214 aminoacids) are encoded by a variable region gene at the NH2-terminus (about110 amino acids) and a kappa or lambda constant region gene at theCOOH-terminus. Full-length immunoglobulin “heavy chains” (about 50 Kd or446 amino acids), are similarly encoded by a variable region gene (about116 amino acids) and one of the other aforementioned constant regiongenes (about 330 amino acids). Heavy chains are classified as gamma, mu,alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM,IgA, IgD and IgE, respectively. Within light and heavy chains, thevariable and constant regions are joined by a “J” region of about 12 ormore amino acids, with the heavy chain also including a “D” region ofabout 10 more amino acids. (See generally, Fundamental Immunology (Paul,W., ed., 2nd ed. Raven Press, N.Y., 1989), Ch. 7 (incorporated herein byreference its disclosure on producing antibodies and antibodyfragments).

An immunoglobulin light or heavy chain variable region consists of a“framework” region interrupted by three hypervariable regions. Thus, theterm “hypervariable region” refers to the amino acid residues of anantibody which are responsible for antigen binding. The hypervariableregion comprises amino acid residues from a “Complementarity DeterminingRegion” or “CDR” (i.e., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3)in the light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102(H3) in the heavy chain variable domain (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)) and/or thoseresidues from a “hypervariable loop” (i.e., residues 26-32 (L1), 50-52(L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1),53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothiaand Lesk, 1987, J. Mol. Biol. 196: 901-917) (both of which areincorporated herein by reference). “Framework Region” or “FR” residuesare those variable domain residues other than the hypervariable regionresidues as herein defined. The sequences of the framework regions ofdifferent light or heavy chains are relatively conserved within aspecies. Thus, a “human framework region” is a framework region that issubstantially identical (about 85% or more, usually 90-95% or more) tothe framework region of a naturally occurring human immunoglobulin. Theframework region of an antibody, that is the combined framework regionsof the constituent light and heavy chains, serves to position and alignthe CDR's. The CDR's are primarily responsible for binding to an epitopeof an antigen.

Accordingly, the term “humanized” immunoglobulin refers to animmunoglobulin comprising a human framework region and one or more CDR'sfrom a non-human (usually a mouse or rat) immunoglobulin. The non-humanimmunoglobulin providing the CDR's is called the “donor” and the humanimmunoglobulin providing the framework is called the “acceptor”.Constant regions need not be present, but if they are, they must besubstantially identical to human immunoglobulin constant regions, i.e.,at least about 85-90%, preferably about 95% or more identical. Hence,all parts of a humanized immunoglobulin, except possibly the CDR's, aresubstantially identical to corresponding parts of natural humanimmunoglobulin sequences. A “humanized antibody” is an antibodycomprising a humanized light chain and a humanized heavy chainimmunoglobulin. For example, a humanized antibody would not encompass atypical chimeric antibody as defined above, e.g., because the entirevariable region of a chimeric antibody is non-human.

The term “recombinant antibodies” means antibodies wherein the aminoacid sequence has been varied from that of a native antibody. Because ofthe relevance of recombinant DNA techniques in the generation ofantibodies, one need not be confined to the sequences of amino acidsfound in natural antibodies; antibodies can be redesigned to obtaindesired characteristics. The possible variations are many and range fromthe changing of just one or a few amino acids to the complete redesignof, for example, the variable or constant region. Changes in theconstant region will, in general, be made in order to improve or altercharacteristics, such as complement fixation, interaction with membranesand other effector functions. Changes in the variable region will bemade in order to improve the antigen binding characteristics.

In addition to antibodies, immunoglobulins may exist in a variety ofother forms including, for example, single-chain or Fv, Fab, and(Fab′)₂, as well as diabodies, linear antibodies, multivalent ormultispecific hybrid antibodies (as described above and in detail in:Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and in singlechains (e.g., Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85,5879-5883 (1988) and Bird et al., Science, 242, 423-426 (1988), whichare incorporated herein by reference for their teachings on antibodyfragments). (See, generally, Hood et al., “Immunology”, Benjamin, N.Y.,2nd ed. (1984), and Hunkapiller and Hood, Nature, 323, 15-16 (1986),which are incorporated herein by reference for antibodies).

As used herein, the terms “single-chain Fv,” “single-chain antibodies,”“Fv” or “scFv” refer to antibody fragments that comprises the variableregions from both the heavy and light chains, but lacks the constantregions, but within a single polypeptide chain. Generally, asingle-chain antibody further comprises a polypeptide linker between theVH and VL domains which enables it to form the desired structure whichwould allow for antigen binding. Single chain antibodies are discussedin detail by Pluckthun in The Pharmacology of Monoclonal Antibodies,vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp.269-315 (1994); see also International Patent Application PublicationNo. WO 88/01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203, thedisclosures of which are incorporated by reference for any purpose. Inspecific embodiments, single-chain antibodies can also be bi-specificand/or humanized.

A “Fab fragment” is comprised of one light chain and the C_(H1) andvariable regions of one heavy chain. The heavy chain of a Fab moleculecannot form a disulfide bond with another heavy chain molecule.

A “Fab′ fragment” contains one light chain and one heavy chain thatcontains more of the constant region, between the C_(H1) and C_(H2)domains, such that an interchain disulfide bond can be formed betweentwo heavy chains to form a F(ab′)₂ molecule.

A “F(ab′) 2 fragment” contains two light chains and two heavy chainscontaining a portion of the constant region between the C_(H1) andC_(H2) domains, such that an interchain disulfide bond is formed betweentwo heavy chains.

Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to ±10%.

All references cited herein are incorporated by reference in theirentirety.

The present invention is based upon the discovery of humanized mouseanti-human IL-31 variable region sequences of antibodies. Use of theseantibodies as antagonists to IL-31 can inhibit inflammation in general,and the symptoms of dermatitis and pruritic diseases in specific. Theinvention provides the use of humanized light chain and heavy chainregions of antibodies that recognize, bind, and/or neutralize the IL-31polypeptide. Such humanized light and heavy chain regions can be fusedto an immunoglobulin constant region, such as for example, IgG4 or IgG1,and expressed in a variety of host cells. The humanized anti-IL-31variable region sequences described herein were generated using aminoacid sequences of mouse anti-human IL-31 light chain and heavy chainvariable regions of monoclonal antibodies previously described in U.S.patent application Ser. No. 11/430,066, filed May 8, 2006 (U.S. PatentPublication No. 2006-02752960), herein incorporated by reference.

Antibodies can comprise antibodies or antibody fragments, comprising orconsisting of a light chain variable region and a heavy chain variableregion, and can be chimeric, humanized, or antibody fragments thatneutralize, inhibit, reduce, prevent or minimize the effects of IL-31 onits receptor. Clinical outcomes of the antibody or antibody fragmentscan be a reduction in inflammatory and autoimmune diseases, such asdermatitis, in particular atopic dermatitis, and pruritic diseases andCrohn's disease, as further described herein. In an embodiment, thedermatitis is atopic dermatitis. In another embodiment the dermatitis isprurigo nodularis. In another embodiment, the dermatitis is eczema. Thereduction may also be a reduction in itch, scratching, or hairloss.

IL-31 is a newly discovered T cell cytokine which, when over-expressedin mice, results in dermatitis-like symptoms. See, Dillon, et al.,Nature Immunol. 5:752-760, 2004. IL-31 is the HUGO name for a cytokinethat has been previously described as Zcyto17rlig in U.S. patentapplication Ser. No. 10/352,554, filed Jan. 21, 2003 (U.S PatentPublication No. 2003-0224487), now U.S. Pat. No. 7,064,186, Sprecher,Cindy et al., 2003, incorporated herein by reference). See also, Dillon,et al., Nature Immunol., supra. The amino acid sequence of human IL-31is shown in SEQ ID NO: 24. Tissue analysis revealed that expression ofmouse IL-31 is found in testis, brain, CD90+ cells, prostate cells,salivary gland and skin.

The heterodimeric receptor for IL-31 was described in U.S. PatentPublicaiton No. 20030224487 as zcytor17 (HUGO name, IL-31RA) which formsa heterodimer with OncostatinM receptor beta (OSMRbeta). IL-31 wasisolated from a cDNA library generated from activated human peripheralblood cells (hPBCs), which were selected for CD3. CD3 is a cell surfacemarker unique to cells of lymphoid origin, particularly T cells.

Inhibition, neutralization, or blocking signal transduction by themolecules comprising a humanized mouse anti-human light chain variabledomain and/or a humanised mouse anti-human heavy chain variable domain,termed “IL-31 antigen binding molecules” or “IL-31 antagonists” herein,can be measured by a number of assays known to one skilled in the art.For example, assays measuring a reduction in proliferation includeassays for reduction of a dye such as AlamarBlue™ (AccuMedInternational, Inc. Westlake, Ohio),3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (Mosman,J. Immunol. Meth. 65: 55-63, 1983); 3, (4,5 dimethylthiazol-2yl)-5-3-carboxymethoxyphenyl-2H-tetrazolium;2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazoliumhydroxide; and cyanoditolyl-tetrazolium chloride (which are commerciallyavailable from Polysciences, Inc., Warrington, Pa.); mitogenesis assays,such as measurement of incorporation of ³H-thymidine; dye exclusionassays using, for example, naphthalene black or trypan blue; dye uptakeusing diacetyl fluorescein; and chromium release. See, in general,Freshney, Culture of Animal Cells: A Manual of Basic Technique, 3rd ed.,Wiley-Liss, 1994, which is incorporated herein by reference. In additionto the above, see published U.S. patent publication number 20030224487,(Sprecher, Cindy et al., 2003) for an example of BaF3 cells expressingIL-31RA and full-length OSMRbeta or as shown in the activity examplesdescribed herein.

Methods for preparing the polynucleotides encoding the antibodiesdescribed herein (including DNA and RNA) are well known in the art.Total RNA can be prepared using guanidinium isothiocyanate extractionfollowed by isolation by centrifugation in a CsCl gradient (Chirgwin etal., Biochemistry 18:52-94, 1979). Poly (A)⁺ RNA is prepared from totalRNA using the method of Aviv and Leder (Proc. Natl. Acad. Sci. USA69:1408-12, 1972). Complementary DNA (cDNA) is prepared from poly(A)⁺RNA using known methods. In the alternative, genomic DNA can beisolated. Polynucleotides encoding IL-31 antibodies are then identifiedand isolated by, for example, hybridization or PCR.

The present invention also includes humanized IL-31 antigen bindingmolecules or IL-31 antagonists that bind functional fragments of IL-31polypeptides and nucleic acid molecules encoding such functionalfragments. A “functional” IL-31 or fragment thereof as defined herein ischaracterized by its proliferative or differentiating activity, by itsability to induce or inhibit specialized cell functions, or by itsability to bind specifically to an anti-IL-31 antibody orIL-31RA/OSMRbeta heterodimers of these receptors (either soluble orimmobilized). Thus, the present invention further provides humanizedIL-31 antigen binding molecules or IL-31 antagonists that bind apolypeptide molecule comprising one or more IL-31 functional fragments.

The present invention also provides humanized IL-31 antigen bindingmolecules or IL-31 antagonists that bind to polypeptide fragments orpeptides comprising an epitope-bearing portion of a IL-31 polypeptide oran immunogenic epitope or antigenic epitope. The binding of theantibodies to these epitopes results in inhibition, blocking,neutralization, and/or reduction in signal transduction of IL-31 on itscognate receptor.

Mouse anti-human IL-31 monoclonal antibodies have been previouslydescribed in co-pending U.S. patent application Ser. No. 11/430,066,filed May 8, 2006 (U.S. Patent Publication No. 2006-02752960). The aminoacid sequences of the variable regions of mouse anti-human IL-31antibodies described in co-pending U.S. patent application Ser. No.11/850,006, filed Sep. 4, 2007 and co-owned PCT application US07/77555,filed Sep. 4, 2007, both of which are incorporated by reference hereinfor the variable region sequences and antibody production methods. Theseamino acid sequences were used as starting material for the humanizedsequences described herein. In specific, the mouse anti-human IL-31antibody sequence was from a hybridoma with the clone number 292.12.3.1.

The activity of the antibodies as described herein can be measured bytheir ability to inhibit, or reduce proliferation using a variety ofassays that measure proliferation of and/or binding to cells expressingthe IL-31RA receptor. Of particular interest are changes inIL-31-dependent cells. Suitable cell lines to be engineered to beIL-31-dependent include the IL-3-dependent BaF3 cell line (Palacios andSteinmetz, Cell 41: 727-734, 1985; Mathey-Prevot et al., Mol. Cell.Biol. 6: 4133-4135, 1986), FDC-P1 (Hapel et al., Blood 64: 786-790,1984), and MO7e (Kiss et al., Leukemia 7: 235-240, 1993). Growthfactor-dependent cell lines can be established according to publishedmethods (e.g. Greenberger et al., Leukemia Res. 8: 363-375, 1984; Dexteret al., in Baum et al. Eds., Experimental Hematology Today, 8th Ann.Mtg. Int. Soc. Exp. Hematol. 1979, 145-156, 1980). The activity of thehumanized anti-IL-31 antibodies or antagonists can also be measured inthe BaF3 proliferation assay, the Biacore assay, or the NHK assaysdescribed herein.

In an embodiment, the immunoglobulin heavy chain constant region domainis selected from the group consisting of the constant region of an α, γ,μ, δ or ε human immunoglobulin heavy chain. Said constant region can bethe constant region of a γ1, γ2, γ3 or γ4 human immunoglobulin heavychain.

In another embodiment, the immunoglobulin light chain constant regiondomain is selected from the group consisting of the constant region of aκ or λ human immunoglobulin light chain

In another embodiment, the heavy constant chain is human γ4, which isstable in solution and has little or no complement activating activity.In another embodiment, the heavy constant chain is human γ1.

The immunoglobulin may be selected from any of the major classes ofimmunoglobulins, including IgA, IgD, IgE, IgG and IgM, and any subclassor isotype, e.g. IgG1, IgG2, IgG3 and IgG4; IgA-1 and IgA-2.

Inhibition the activity of IL-31 can be measured by a number of assays.In addition to those assays disclosed herein, samples can be tested forinhibition of IL-31 activity within a variety of assays designed tomeasure receptor binding, the stimulation/inhibition of IL-31-dependentcellular responses or proliferation of IL-31RA receptor-expressingcells.

A IL-31-binding polypeptide, including IL-31 antigen binding moleculesor IL-31 antagonists can also be used for purification of ligand. Thepolypeptide is immobilized on a solid support, such as beads of agarose,cross-linked agarose, glass, cellulosic resins, silica-based resins,polystyrene, cross-linked polyacrylamide, or like materials that arestable under the conditions of use. Methods for linking polypeptides tosolid supports are known in the art, and include amine chemistry,cyanogen bromide activation, N-hydroxysuccinimide activation, epoxideactivation, sulfhydryl activation, and hydrazide activation. Theresulting medium will generally be configured in the form of a column,and fluids containing ligand are passed through the column one or moretimes to allow ligand to bind to the receptor polypeptide. The ligand isthen eluted using changes in salt concentration, chaotropic agents(guanidine HCl), or pH to disrupt ligand-receptor binding.

Humanized IL-31 antigen binding molecules or IL-31 antagonists areconsidered to be specifically binding if: 1) they exhibit a thresholdlevel of binding activity, and 2) they do not significantly cross-reactwith related polypeptide molecules. A threshold level of binding isdetermined if IL-31 antigen binding molecules or IL-31 antagonistsherein bind to a IL-31 polypeptide, peptide or epitope with an affinityat least 10-fold greater than the binding affinity to control(non-IL-31) polypeptide. It is preferred that the antibodies exhibit abinding affinity (K_(a)) of 10⁶ M⁻¹ or greater, preferably 10⁷ M⁻¹ orgreater, more preferably 10⁸ M⁻¹ or greater, and most preferably 10⁹ M⁻¹or greater. The binding affinity of humanized IL-31 antigen bindingmolecules or IL-31 antagonists can be readily determined by one ofordinary skill in the art, for example, by Scatchard analysis(Scatchard, G., Ann. NY Acad. Sci. 51: 660-672, 1949).

Whether IL-31 antigen binding molecules or IL-31 antagonists do notsignificantly cross-react with related polypeptide molecules is shown,for example, by the IL-31 antigen binding molecules or IL-31 antagonistsdetecting IL-31 polypeptide but not known related polypeptides using astandard Western blot analysis (Ausubel et al., ibid.). Screening canalso be done using non-human IL-31, and IL-31 mutant polypeptides.Moreover, IL-31 antigen binding molecules or IL-31 antagonists can be“screened against” known related polypeptides, to isolate a populationthat specifically binds to the IL-31 polypeptides. For example, IL-31antigen binding molecules or IL-31 antagonists are adsorbed to relatedpolypeptides adhered to insoluble matrix; IL-31 antigen bindingmolecules or IL-31 antagonists specific to IL-31 will flow through thematrix under the proper buffer conditions. Antibodies: A LaboratoryManual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press,1988; Current Protocols in Immunology, Cooligan, et al. (eds.), NationalInstitutes of Health, John Wiley and Sons, Inc., 1995.

Humanized IL-31 antigen binding molecules or IL-31 antagonists arecharacterized for their ability to block, inhibit, prevent, or reducereceptor binding when grown in the presence of the purified recombinantproteins human IL-31. For example, the humanized IL-31 antigen bindingmolecules or IL-31 antagonists can be characterized in a number of waysincluding binning (i.e, determining if each antibody could inhibit thebinding of any other binding), relative affinity, and neutralization.

The humanized IL-31 antigen binding molecules or IL-31 antagonists ofthe invention are shown in Table 1, below, and in FIG. 1.

In Table 1, below, for the clones having the designation of “12VH1”(i.e., clones 7, 9, 10, 13-18, and 26-36): the term “fback” meansLC(back)+HC(fback); the term “LC(back)” means LC(G66R, G68E, D70Q, F71Y,T72S); and the term “HC(back)” means HC(F29L, M69L, R71A, T73K, V78A,R94F). Similarly, in Table 1, below for the clones having thedesignation of “12VH5” (i.e., clones 8, 11 and 21-24): the term “fback”means LC(back)+HC(fback); the term “LC(back)” means LC(G66R, G68E, D70Q,F71Y, T72S); and the term “HC(back)” means HC(G24A, S28T, F29L, I69L,S70T, R94F).

TABLE 1 Humanized 292.12.3.1 constructs Heavy Light Chain Chain CloneSEQ ID SEQ ID Number Mutation Description NO: NO: 7 12VH1-gly + fback 2526 8 12VH5-gly + fback 27 26 9 12VH1-gly + HC(fback) 25 28 1012VH1-gly + LC(fback) 29 26 11 12VH5-gly + HC(fback) 27 28 1312VH1-gly + HC(F29L, M69L, R71A, T73K, 30 26 V78A) + LC(fback) 1412VH1-gly + HC(M69L, R71A, T73K, V78A, 31 26 R94F) + LC(fback) 1612VH1-gly + HC(fback) + LC(G66R, G68E, D70Q, T72S) 25 32 17 12VH1-gly +HC(fback) + LC(D70Q, F71Y, T72S) 25 33 18 12VH1-gly + HC(fback) +LC(G66R, G68E, F71Y) 25 34 21 12VH5-gly + HC(G24A, S28T, F29L, R94F) +LC(fback) 35 26 22 12VH5-gly + HC(fback) + LC(G66R, G68E, D70Q, T72S) 2732 23 12VH5-gly + HC(fback) + LC(D70Q, F71Y, T72S) 27 33 24 12VH5-gly +HC(fback) + LC(G66R, G68E, F71Y) 27 34 25 12VH1-gly + HC(F29L, R94F) +LC(F71Y) 36 37 26 12VH1-gly + HC(fback) + LC(F71Y) 25 37 27 12VH1-gly +HC(F29L, M69L, R94F) + LC(F71Y) 38 37 28 12VH1-gly + HC(F29L, R71A,R94F) + LC(F71Y) 39 37 29 12VH1-gly + HC(F29L, T73K, R94F) + LC(F71Y) 4037 30 12VH1-gly + HC(F29L, V78A, R94F) + LC(F71Y) 41 37 31 12VH1-gly +HC(F29L, M69L, R71A, V78A, R94F) + LC(F71Y) 42 37 32 12VH1-gly +HC(F29L, R71A, V78A, R94F) + LC(F71Y) 43 37 33 12VH1(germ)-gly + fback44 26 34 12VH1-gly + HC(F29L, R94F) + LC(fback) 36 28 35 12VH1-gly +HC(F29L, R94F) 36 28 36 12VH1(germ)-gly + HC(F29L, T73K, R94F) +LC(F71Y) 45 37

The antibodies disclosed herein may be produced by any technique knownper se in the art, such as by recombinant technologies, chemicalsynthesis, cloning, ligations, or combinations thereof. In a embodiment,the antibodies of the present invention are produced by recombinanttechnologies, e.g., by expression of a corresponding nucleic acid in asuitable host cell. The polypeptide produced may be glycosylated or not,or may contain other post-translational modifications depending on thehost cell type used. Many books and reviews provide teachings on how toclone and produce recombinant proteins using vectors and prokaryotic oreukaryotic host cells, such as some titles in the series “A PracticalApproach” published by Oxford University Press (“DNA Cloning 2:Expression Systems”, 1995; “DNA Cloning 4: Mammalian Systems”, 1996;“Protein Expression”, 1999; “Protein Purification Techniques”, 2001).

A further object of the present invention is therefore an isolatednucleic acid molecule encoding any of the antibodies here above or belowdescribed, or a complementary strand or degenerate sequence thereof. Inthis regard, the term “nucleic acid molecule” encompasses all differenttypes of nucleic acids, including without limitation deoxyribonucleicacids (e.g., DNA, cDNA, gDNA, synthetic DNA, etc.), ribonucleic acids(e.g., RNA, mRNA, etc.) and peptide nucleic acids (PNA). In a preferredembodiment, the nucleic acid molecule is a DNA molecule, such as adouble-stranded DNA molecule or a cDNA molecule. The term “isolated”means nucleic acid molecules that have been identified and separatedfrom at least one contaminant nucleic acid molecule with which it isordinarily associated in the natural source. An isolated nucleic acidmolecule is other than in the form or setting in which it is found innature. Isolated nucleic acid molecules therefore are distinguished fromthe specific nucleic acid molecule as it exists in natural cells. Adegenerate sequence designates any nucleotide sequence encoding the sameamino acid sequence as a reference nucleotide sequence, but comprising adistinct nucleotide sequence as a result of the genetic code degeneracy.

A further object of this invention is a vector comprising DNA encodingany of the above or below described antibodies. The vector may be anycloning or expression vector, integrative or autonomously replicating,functional in any prokaryotic or eukaryotic cell. In particular, thevector may be a plasmid, cosmid, virus, phage, episome, artificialchromosome, and the like. The vector may comprise the coding sequencesfor both the heavy and light chain, or either of the light and heavychain coding sequences. Should the vector comprise coding sequences forboth heavy and light chains, the heavy and light chains may each beoperably linked to a promoter. The promoter may be the same or differentfor the heavy and light chain. The heavy and light chain may also beoperably linked to one single promoter, in this case the codingsequences for the heavy and light chains may preferably be separated byan internal ribosomal entry site (IRES). Suitable promoters foreukaryotic gene expression are, for example, promoters derived fromviral genes such as the murine or human cytomegalovirus (CMV) or therous sarcoma virus (RSV) promoter, which are well known to the personskilled in the art. The vector may comprise regulatory elements, such asa promoter, terminator, enhancer, selection marker, origin ofreplication, etc. Specific examples of such vectors include prokaryoticplasmids, such as pBR, pUC or pcDNA plasmids; viral vectors, includingretroviral, adenoviral or AAV vectors; bacteriophages; baculoviruses;BAC or YAC, etc., as will be discussed below. The appropriate nucleicacid sequence may be inserted into the vector by a variety ofprocedures. In general, DNA is inserted into an appropriate restrictionendonuclease site(s) using techniques known in the art. Construction ofsuitable vectors containing one or more of these components employsstandard ligation techniques which are known to the skilled artisan.

A further aspect of the present invention is a recombinant host cell,wherein said cell comprises a nucleic acid molecule or a vector asdefined above. The host cell may be a prokaryotic or eukaryotic cell.Examples of prokaryotic cells include bacteria, such as E. coli.Examples of eucaryotic cells are yeast cells, plant cells, mammaliancells and insect cells including any primary cell culture or establishedcell line (e.g., 3T3, Véro, HEK293, TNS, etc.). Suitable host cells forthe expression of glycosylated proteins are derived from multicellularorganisms. Examples of invertebrate cells include insect cells such asDrosophila S2 and Spodoptera 5f9, as well as plant cells. Examples ofuseful mammalian host cell lines include Chinese hamster ovary (CHO) andCOS cells. More specific examples include monkey kidney CV1 linetransformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line(293 or 293 cells subcloned for growth in suspension culture, Graham etal., J. Gen Virol., 36:59 (1977)); Chinese hamster ovary cells/-DHFR(CHO, Urlaub and Chasin, Proc. Natl, Acad. Sci. USA, 77:4216 (1980));mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 (1980));human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB8065); and mouse mammary tumor (MMT 060562, ATCC CCL51). Particularlypreferred mammalian cells of the present invention are CHO cells.Further particularly preferred mammalian cells suitable for expressionof the antibodies of the invention are murine cells, such as mousemyeloma (N50) cells.

As disclosed here above, the antibodies of the present invention may beproduced by any technique known per se in the art, such as byrecombinant technologies, chemical synthesis, cloning, ligations, orcombinations thereof. In a particular embodiment, the soluble receptorsare produced by recombinant technologies, e.g., by expression of acorresponding nucleic acid in a suitable host cell. Another object ofthis invention is therefore a method of producing an antibody of thepresent invention, the method comprising culturing a recombinant hostcell of the invention under conditions allowing expression of thenucleic acid molecule, and recovering the polypeptide produced. Themethod of producing of the present invention may further comprise thestep of formulating the polypeptide into a pharmaceutical composition.The polypeptide produced may be glycosylated or not, or may containother post-translational modifications depending on the host cell typeused. Many books and reviews provide teachings on how to clone andproduce recombinant proteins using vectors and prokaryotic or eukaryotichost cells, such as some titles in the series “A Practical Approach”published by Oxford University Press (“DNA Cloning 2: ExpressionSystems”, 1995; “DNA Cloning 4: Mammalian Systems”, 1996; “ProteinExpression”, 1999; “Protein Purification Techniques”, 2001).

The vectors to be used in the method of producing an antibody accordingto the present invention can be episomal or non-/homologouslyintegrating vectors, which can be introduced into the appropriate hostcells by any suitable means (transformation, transfection, conjugation,protoplast fusion, electroporation, calcium phosphate-precipitation,direct microinjection, etc.). Factors of importance in selecting aparticular plasmid, viral or retroviral vector include: the ease withwhich recipient cells that contain the vector may be recognized andselected from those recipient cells which do not contain the vector; thenumber of copies of the vector which are desired in a particular host;and whether it is desirable to be able to “shuttle” the vector betweenhost cells of different species. The vectors should allow the expressionof the polypeptide or fusion proteins of the invention in prokaryotic oreukaryotic host cells, under the control of appropriate transcriptionalinitiation/termination regulatory sequences, which are chosen to beconstitutively active or inducible in said cell. A cell linesubstantially enriched in such cells can be then isolated to provide astable cell line.

Host cells are transfected or transformed with expression or cloningvectors described herein for protein production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences. The culture conditions, such as media, temperature,pH and the like, can be selected by the skilled artisan without undueexperimentation. In general, principles, protocols, and practicaltechniques for maximizing the productivity of cell cultures can be foundin Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed.(IRL Press, 1991) and Sambrook et al., supra.

For eukaryotic host cells (e.g. yeasts, insect or mammalian cells),different transcriptional and translational regulatory sequences may beemployed, depending on the nature of the host. They may be derived formviral sources, such as adenovirus, papilloma virus, Simian virus or thelike, where the regulatory signals are associated with a particular genewhich has a high level of expression. Examples are the TK promoter ofthe Herpes virus, the SV40 early promoter, the yeast gal4 gene promoter,etc. Transcriptional initiation regulatory signals may be selected whichallow for repression and activation, so that expression of the genes canbe modulated. The cells which have been stably transformed by theintroduced DNA can be selected by also introducing one or more markerswhich allow for selection of host cells which contain the expressionvector. The marker may also provide for phototrophy to an auxotrophichost, biocide resistance, e.g. antibiotics, or heavy metals such ascopper, or the like. The selectable marker gene can be either directlylinked to the DNA sequences to be expressed (e.g., on the same vector),or introduced into the same cell by co-transfection. Additional elementsmay also be needed for optimal synthesis of proteins of the invention.

Suitable prokaryotic cells include bacteria (such as Bacillus subtilisor E. coli) transformed with a recombinant bacteriophage, plasmid orcosmid DNA expression vector. Such cells typically produce proteinscomprising a N-terminal Methionine residue. Preferred cells to be usedin the present invention are eukaryotic host cells, e g mammalian cells,such as human, monkey, mouse, and Chinese Hamster Ovary (CHO) cells,because they provide post-translational modifications to proteinmolecules, including correct folding or glycosylation at correct sites.Examples of suitable mammalian host cells include African green monkeykidney cells (Vero; ATCC CRL 1587), human embryonic kidney cells(293-HEK; ATCC CRL 1573), baby hamster kidney cells (BHK-21, BHK-570;ATCC CRL 8544, ATCC CRL 10314), canine kidney cells (MDCK; ATCC CCL 34),Chinese hamster ovary cells (CHO-K1; ATCC CCL61; CHO DG44 (Chasin etal., Som. Cell. Molec. Genet. 12:555, 1986)), rat pituitary cells (GH1;ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E;ATCC CRL 1548), SV40-transformed monkey kidney cells (COS-1; ATCC CRL1650), Bowes melanoma and human hepatocellular carcinoma (for exampleHep G2), murine embryonic cells (NIH-3T3; ATCC CRL 1658) and a number ofother cell lines. Alternative eukaryotic host cells are yeast cells(e.g., Saccharomyces, Kluyveromyces, etc.) transformed with yeastexpression vectors. Also yeast cells can carry out post-translationalpeptide modifications including glycosylation. A number of recombinantDNA strategies exist which utilize strong promoter sequences and highcopy number of plasmids that can be utilized for production of thedesired proteins in yeast. Yeast cells recognize leader sequences incloned mammalian gene products and secrete polypeptides bearing leadersequences (i.e., pre-peptides).

For long-term, high-yield production of a recombinant polypeptide,stable expression is preferred. For example, cell lines which stablyexpress the polypeptide of interest may be transformed using expressionvectors which may contain viral origins of replication and/or endogenousexpression elements and a selectable marker gene on the same or on aseparate vector. Following the introduction of the vector, cells may beallowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells that successfully express the introduced sequences.Resistant clones of stably transformed cells may be proliferated usingtissue culture techniques appropriate to the cell type. A cell linesubstantially enriched in such cells can be then isolated to provide astable cell line.

A particularly preferred method of high-yield production of arecombinant polypeptide of the present invention is through the use ofdihydrofolate reductase (DHFR) amplification in DHFR-deficient CHOcells, by the use of successively increasing levels of methotrexate asdescribed in U.S. Pat. No. 4,889,803. The polypeptide obtained may be ina glycosylated form.

Antibodies disclosed herein can also be expressed in other eukaryoticcells, such as avian, fungal, insect, yeast, or plant cells. Thebaculovirus system provides an efficient means to introduce cloned genesinto insect cells. The materials for baculovirus/insect cell expressionsystems are commercially available in kit form from, inter alia,Invitrogen.

In addition to recombinant DNA technologies, the antibodies of thisinvention may be prepared by chemical synthesis technologies. Examplesof chemical synthesis technologies are solid phase synthesis and liquidphase synthesis. As a solid phase synthesis, for example, the amino acidcorresponding to the carboxy-terminus of the polypeptide to besynthesised is bound to a support which is insoluble in organic solventsand, by alternate repetition of reactions (e.g., by sequentialcondensation of amino acids with their amino groups and side chainfunctional groups protected with appropriate protective groups), thepolypeptide chain is extended. Solid phase synthesis methods are largelyclassified by the tBoc method and the Fmoc method, depending on the typeof protective group used. Totally synthetic proteins are disclosed inthe literature (Brown A et al., 1996).

The antibodies of the present invention can be produced, formulated,administered, or generically used in other alternative forms that can bepreferred according to the desired method of use and/or production. Theproteins of the invention can be post-translationally modified, forexample by glycosylation. The polypeptides or proteins of the inventioncan be provided in isolated (or purified) biologically active form, oras precursors, derivatives and/or salts thereof.

Useful conjugates or complexes can also be generated for improving theagents in terms of drug delivery efficacy. For this purpose, theantibodies described herein can be in the form of active conjugates orcomplex with molecules such as polyethylene glycol and other natural orsynthetic polymers (Harris J M and Chess R B, 2003; Greenwald R B etal., 2003; Pillai O and Panchagnula R, 2001). In this regard, thepresent invention contemplates chemically modified antibodies asdisclosed herein, in which the antibody is linked with a polymer.Typically, the polymer is water soluble so that the conjugate does notprecipitate in an aqueous environment, such as a physiologicalenvironment. An example of a suitable polymer is one that has beenmodified to have a single reactive group, such as an active ester foracylation, or an aldehyde for alkylation. In this way, the degree ofpolymerization can be controlled. An example of a reactive aldehyde ispolyethylene glycol propionaldehyde, ormono-(C1-C10) alkoxy, or aryloxyderivatives thereof (see, for example, Harris, et al., U.S. Pat. No.5,252,714). The polymer may be branched or unbranched. Moreover, amixture of polymers can be used to produce the conjugates. Theconjugates used for therapy can comprise pharmaceutically acceptablewater-soluble polymer moieties. Suitable water-soluble polymers includepolyethylene glycol (PEG), monomethoxy-PEG, mono-(C1-C10) alkoxy-PEG,aryloxy-PEG, poly-(N-vinyl pyrrolidone) PEG, tresyl monomethoxy PEG, PEGpropionaldehyde, bis-succinimidyl carbonate PEG, propylene glycolhomopolymers, a polypropyleneoxide/ethylene oxide co-polymer,polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, dextran,cellulose, or other carbohydrate-based polymers. Suitable PEG may have amolecular weight from about 600 to about 60,000, including, for example,5,000, 12,000, 20,000 and 25,000. A conjugate can also comprise amixture of such water-soluble polymers.

Examples of conjugates comprise any of the antibody disclosed here aboveand a polyallcyl oxide moiety attached to the N-terminus of said solublereceptor. PEG is one suitable polyalkyl oxide. As an illustration, anyof the antibody disclosed herein can be modified with PEG, a processknown as “PEGylation.” PEGylation can be carried out by any of thePEGylation reactions known in the art (see, for example, EP 0 154 316,Delgado et al., Critical Reviews in Therapeutic Drug Carrier Systems 9:249 (1992), Duncan and Spreafico, Clin. Pharmacokinet. 27: 290 (1994),and Francis et al., Int J Hematol 68: 1 (1998)). For example, PEGylationcan be performed by an acylation reaction or by an alkylation reactionwith a reactive polyethylene glycol molecule. In an alternativeapproach, conjugates are formed by condensing activated PEG, in which aterminal hydroxy or amino group of PEG has been replaced by an activatedlinker (see, for example, Karasiewicz et al., U.S. Pat. No. 5,382,657).Preferably, all these modifications do not affect significantly theability of the antibody to bind human IL-31.

The antibodies described herein may comprise an additional N-terminalamino acid residue, preferably a methionine. Indeed, depending on theexpression system and conditions, polypeptides may be expressed in arecombinant host cell with a starting Methionine. This additional aminoacid may then be either maintained in the resulting recombinant protein,or eliminated by means of an exopeptidase, such as MethionineAminopeptidase, according to methods disclosed in the literature (VanValkenburgh H A and Kahn R A, Methods Enzymol. (2002) 344:186-93;Ben-Bassat A, Bioprocess Technol. (1991) 12:147-59).

As it will be shown in the example part of the present document, mouseanti-human anti-IL-31 antibodies may include potential glycosylationsites in their variable domains. For example, clone 292.12.3.1 bears aglycosylation site in the framework region FR1 of the heavy chainvariable domain. The inventors of the present invention have now foundthat said glycosylation site is not necessary to keep a good bindingaffinity for human IL-31. Moreover, abolishing this glycosylation site(e.g. by mutation) has a positive effect on the thermostability of theantibody. Therefore in an embodiment of the present invention, theantibodies as disclosed herein do not have glycosylation site in theirvariable domains.

IL-31 antigen binding molecules or IL-31 antagonists generated by themethods described herein can be tested for neutralization by a varietyof methods. For example the luciferase assay as described in publishedU.S. patent application (See publication number 20030224487, Sprecher,Cindy et al., 2003) can be used. In addition neutralization can betested by measuring a decrease in the production of pro-inflammatorychemokines such as TARC and MDC from keratinocyte cultures in thepresence of ligand and the monoclonal antibody. Neutralization can alsobe measured by the in vivo and in vitro assays described herein.

Reduction of TARC and MDC in response to the humanized anti-IL-31antibodies and antagonists described herein can be measured in an ADmouse model as follows:

Method I) Six-week old male NC/Nga mice (CRL Japan) are sensitizedintradermally with 50 μg dust mite extract (D. pteronyssinus, IndoorBiotechnologies) three times a week on the back and scored for AD-likelesions. After 5 weeks of sensitization the mice are euthanized and theright ears were excised and placed into a single well of a 48-wellculture dish (Corning) supplemented with RPMI+2% FBS (GIBCO Invitrogen).Plates are placed in 5% CO2 humidity controlled incubators. Supernatantsare collected after 24 hours and frozen at −20° C. until furtheranalysis.

Method II) Twelve-week old female NC/Nga mice (CRL Japan) are sensitizedintradermally with 10 μg SEB (Toxin Technology) in the ear and on theback three times per week. The mice are scored for AD-like lesions.After 5 weeks of sensitization the mice are euthanized and 6 mm biopsypunches were taken from the injected ear of each mouse and placed into asingle well of a 48-well culture dish supplemented with RPMI+2% FBS.Plates were placed in 5% CO2 humidity controlled incubators.Supernatants are collected after 24 hours and frozen at −20° C. untilfurther analysis.

Groups of mice in both studies are treated with humanized IL-31 antigenbinding molecules or the humanized IL-31 antibodies or antagonistsintraperitoneally two times each week starting after 1 to 2 weeks ofsensitization. TARC and MDC concentrations in the 24-hour supernatantsamples are measured by conventional ELISA (R&D Systems).

In one embodiment, the humanized IL-31 antigen binding molecules orIL-31 antagonists of the present invention include, but are not limitedto, Fab, Fab′ and F(ab)₂, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv) and fragments comprising eithera VL or VH domain. Antigen-binding antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) (i.e., SEQID NOs: 25-45) alone or in combination with the entirety or a portion ofthe following: hinge region, C_(H1), C_(H2), and C_(H3) domains. Alsoincluded in the invention are antigen-binding fragments also comprisingany combination of variable region(s) with a hinge region, C_(H1),C_(H2), and C_(H3) domains. In another embodiment, the heavy chainvariable region of any of SEQ ID NO:s 25; 27; 29; 30; 31; 35; 36; 38;39; 40; 41; 42; 43; 44, and 45 can be combined with the light chainvariable region of any of SEQ ID NOs: 26; 28; 32; 33; 34; and 37.

In another embodiment, the invention provides an isolated antibody orantibody fragment that binds to human IL-31, comprising a humanizedheavy chain variable domain and a humanized light chain variable domainwherein the humanized heavy chain variable domain and a humanized lightchain variable domain are selected from the group consisting of: a) ahumanized heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO: 25 and a humanized light chain variable domain comprisingthe amino acid sequence of SEQ ID NO: 26; b) a humanized heavy chainvariable domain comprising the amino acid sequence of SEQ ID NO: 27 anda humanized light chain variable domain comprising the amino acidsequence of SEQ ID NO:26; c) a humanized heavy chain variable domaincomprising the amino acid sequence of SEQ ID NO: 25 and a humanizedlight chain variable domain comprising the amino acid sequence of SEQ IDNO: 28; d) a humanized heavy chain variable domain comprising the aminoacid sequence of SEQ ID NO: 29 and a humanized light chain variabledomain comprising the amino acid sequence of SEQ ID NO: 26; e) ahumanized heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO: 27 and a humanized light chain variable domain comprisingthe amino acid sequence of SEQ ID NO: 28; f) a humanized heavy chainvariable domain comprising the amino acid sequence of SEQ ID NO: 30 anda humanized light chain variable domain comprising the amino acidsequence of SEQ ID NO: 26; g) a humanized heavy chain variable domaincomprising the amino acid sequence of SEQ ID NO: 31 and a humanizedlight chain variable domain comprising the amino acid sequence of SEQ IDNO: 26; h) a humanized heavy chain variable domain comprising the aminoacid sequence of SEQ ID NO: 25 and a humanized light chain variabledomain comprising the amino acid sequence of SEQ ID NO: 32; i) ahumanized heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO: 25 and a humanized light chain variable domain comprisingthe amino acid sequence of SEQ ID NO: 33; j) a humanized heavy chainvariable domain comprising the amino acid sequence of SEQ ID NO: 25 anda humanized light chain variable domain comprising the amino acidsequence of SEQ ID NO: 34; k) a humanized heavy chain variable domaincomprising the amino acid sequence of SEQ ID NO: 35 and a humanizedlight chain variable domain comprising the amino acid sequence of SEQ IDNO: 26; l) a humanized heavy chain variable domain comprising the aminoacid sequence of SEQ ID NO: 27 and a humanized light chain variabledomain comprising the amino acid sequence of SEQ ID NO: 32; m) ahumanized heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO: 27 and a humanized light chain variable domain comprisingthe amino acid sequence of SEQ ID NO: 33; n) a humanized heavy chainvariable domain comprising the amino acid sequence of SEQ ID NO: 27 anda humanized light chain variable domain comprising the amino acidsequence of SEQ ID NO: 34; o) a humanized heavy chain variable domaincomprising the amino acid sequence of SEQ ID NO: 36 and a humanizedlight chain variable domain comprising the amino acid sequence of SEQ IDNO: 37; p) a humanized heavy chain variable domain comprising the aminoacid sequence of SEQ ID NO: 25 and a humanized light chain variabledomain comprising the amino acid sequence of SEQ ID NO: 37; q) ahumanized heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO: 38 and a humanized light chain variable domain comprisingthe amino acid sequence of SEQ ID NO: 37; r) a humanized heavy chainvariable domain comprising the amino acid sequence of SEQ ID NO: 39 anda humanized light chain variable domain comprising the amino acidsequence of SEQ ID NO: 37; s) a humanized heavy chain variable domaincomprising the amino acid sequence of SEQ ID NO: 40 and a humanizedlight chain variable domain comprising the amino acid sequence of SEQ IDNO: 37; t) a humanized heavy chain variable domain comprising the aminoacid sequence of SEQ ID NO: 41 and a humanized light chain variabledomain comprising the amino acid sequence of SEQ ID NO: 37; u) ahumanized heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO: 42 and a humanized light chain variable domain comprisingthe amino acid sequence of SEQ ID NO: 37; v) a humanized heavy chainvariable domain comprising the amino acid sequence of SEQ ID NO: 43 anda humanized light chain variable domain comprising the amino acidsequence of SEQ ID NO: 37; w) a humanized heavy chain variable domaincomprising the amino acid sequence of SEQ ID NO: 44 and a humanizedlight chain variable domain comprising the amino acid sequence of SEQ IDNO: 26; x) a humanized heavy chain variable domain comprising the aminoacid sequence of SEQ ID NO: 36 and a humanized light chain variabledomain comprising the amino acid sequence of SEQ ID NO: 28; y) ahumanized heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO: 36 and a humanized light chain variable domain comprisingthe amino acid sequence of SEQ ID NO: 28; and z) a humanized heavy chainvariable domain comprising the amino acid sequence of SEQ ID NO: 45 anda humanized light chain variable domain comprising the amino acidsequence of SEQ ID NO: 37.

In another embodiment, the invention provides an isolated antibody orantibody fragment that binds to human IL-31, comprising a humanizedheavy chain variable domain and a humanized light chain variable domainwherein the humanized heavy chain variable domain and a humanized lightchain variable domain are selected from the group consisting of: a) ahumanized heavy chain variable domain having at least 90% sequenceidentity to the amino acid sequence of SEQ ID NO: 25 and a humanizedlight chain variable domain having at least 90% sequence identity to theamino acid sequence of SEQ ID NO: 26; b) a humanized heavy chainvariable domain having at least 90% sequence identity to the amino acidsequence of SEQ ID NO: 27 and a humanized light chain variable domainhaving at least 90% sequence identity to the amino acid sequence of SEQID NO: 26; c) a humanized heavy chain variable domain having at least90% sequence identity to the amino acid sequence of SEQ ID NO: 25 and ahumanized light chain variable domain having at least 90% sequenceidentity to the amino acid sequence of SEQ ID NO: 28; d) a humanizedheavy chain variable domain having at least 90% sequence identity to theamino acid sequence of SEQ ID NO: 29 and a humanized light chainvariable domain having at least 90% sequence identity to the amino acidsequence of SEQ ID NO: 26; e) a humanized heavy chain variable domainhaving at least 90% sequence identity to the amino acid sequence of SEQID NO: 27 and a humanized light chain variable domain having at least90% sequence identity to the amino acid sequence of SEQ ID NO: 28; f) ahumanized heavy chain variable domain having at least 90% sequenceidentity to the amino acid sequence of SEQ ID NO: 30 and a humanizedlight chain variable domain having at least 90% sequence identity to theamino acid sequence of SEQ ID NO: 26; g) a humanized heavy chainvariable domain having at least 90% sequence identity to the amino acidsequence of SEQ ID NO: 31 and a humanized light chain variable domainhaving at least 90% sequence identity to the amino acid sequence of SEQID NO: 26; h) a humanized heavy chain variable domain having at least90% sequence identity to the amino acid sequence of SEQ ID NO: 25 and ahumanized light chain variable domain having at least 90% sequenceidentity to the amino acid sequence of SEQ ID NO: 32; i) a humanizedheavy chain variable domain having at least 90% sequence identity to theamino acid sequence of SEQ ID NO: 25 and a humanized light chainvariable domain having at least 90% sequence identity to the amino acidsequence of SEQ ID NO: 33; j) a humanized heavy chain variable domainhaving at least 90% sequence identity to the amino acid sequence of SEQID NO: 25 and a humanized light chain variable domain having at least90% sequence identity to the amino acid sequence of SEQ ID NO: 34; k) ahumanized heavy chain variable domain having at least 90% sequenceidentity to the amino acid sequence of SEQ ID NO: 35 and a humanizedlight chain variable domain having at least 90% sequence identity to theamino acid sequence of SEQ ID NO: 26; l) a humanized heavy chainvariable domain having at least 90% sequence identity to the amino acidsequence of SEQ ID NO: 27 and a humanized light chain variable domainhaving at least 90% sequence identity to the amino acid sequence of SEQID NO: 32; m) a humanized heavy chain variable domain having at least90% sequence identity to the amino acid sequence of SEQ ID NO: 27 and ahumanized light chain variable domain having at least 90% sequenceidentity to the amino acid sequence of SEQ ID NO: 33; n) a humanizedheavy chain variable domain having at least 90% sequence identity to theamino acid sequence of SEQ ID NO: 27 and a humanized light chainvariable domain having at least 90% sequence identity to the amino acidsequence of SEQ ID NO: 34; o) a humanized heavy chain variable domainhaving at least 90% sequence identity to the amino acid sequence of SEQID NO: 36 and a humanized light chain variable domain having at least90% sequence identity to the amino acid sequence of SEQ ID NO: 37; p) ahumanized heavy chain variable domain having at least 90% sequenceidentity to the amino acid sequence of SEQ ID NO: 25 and a humanizedlight chain variable domain comprising the amino acid sequence of SEQ IDNO: 37; q) a humanized heavy chain variable domain comprising the aminoacid sequence of SEQ ID NO: 38 and a humanized light chain variabledomain comprising the amino acid sequence of SEQ ID NO: 37; r) ahumanized heavy chain variable domain comprising the amino acid sequenceof SEQ ID NO: 39 and a humanized light chain variable domain comprisingthe amino acid sequence of SEQ ID NO: 37; s) a humanized heavy chainvariable domain having at least 90% sequence identity to the amino acidsequence of SEQ ID NO: 40 and a humanized light chain variable domainhaving at least 90% sequence identity to the amino acid sequence of SEQID NO: 37; t) a humanized heavy chain variable domain having at least90% sequence identity to the amino acid sequence of SEQ ID NO: 41 and ahumanized light chain variable domain having at least 90% sequenceidentity to the amino acid sequence of SEQ ID NO: 37; u) a humanizedheavy chain variable domain having at least 90% sequence identity to theamino acid sequence of SEQ ID NO: 42 and a humanized light chainvariable domain having at least 90% sequence identity to the amino acidsequence of SEQ ID NO: 37; v) a humanized heavy chain variable domainhaving at least 90% sequence identity to the amino acid sequence of SEQID NO: 43 and a humanized light chain variable domain having at least90% sequence identity to the amino acid sequence of SEQ ID NO: 37; w) ahumanized heavy chain variable domain having at least 90% sequenceidentity to the amino acid sequence of SEQ ID NO: 44 and a humanizedlight chain variable domain having at least 90% sequence identity to theamino acid sequence of SEQ ID NO: 26; x) a humanized heavy chainvariable domain having at least 90% sequence identity to the amino acidsequence of SEQ ID NO: 36 and a humanized light chain variable domainhaving at least 90% sequence identity to the amino acid sequence of SEQID NO: 28; y) a humanized heavy chain variable domain having at least90% sequence identity to the amino acid sequence of SEQ ID NO: 36 and ahumanized light chain variable domain having at least 90% sequenceidentity to the amino acid sequence of SEQ ID NO: 28; and z) a humanizedheavy chain variable domain having at least 90% sequence identity to theamino acid sequence of SEQ ID NO: 45 and a humanized light chainvariable domain having at least 90% sequence identity to the amino acidsequence of SEQ ID NO: 37.

In an aspect the invention provides an isolated antibody selected fromthe group consisting of: a) an antibody comprising a light chainconsisting of amino acids sequence SEQ ID NO: 46 and a heavy chainconsisting of amino acid sequence SEQ ID NO: 47; b) an antibodycomprising a light chain consisting of amino acids sequence SEQ ID NO:48 and a heavy chain consisting of amino acid sequence SEQ ID NO: 49;and c) an antibody comprising a light chain consisting of amino acidssequence SEQ ID NO: 50 and a heavy chain consisting of amino acidsequence SEQ ID NO: 51.

The present invention also includes recombinant humanized IL-31 antigenbinding molecules or IL-31 antagonists that are functionally equivalentto those described above. Modified humanized IL-31 antigen bindingmolecules or IL-31 antagonists providing improved stability and/ortherapeutic efficacy are also included. Examples of modified antibodiesinclude those with conservative substitutions of amino acid residues,and one or more deletions or additions of amino acids which do notsignificantly deleteriously alter the antigen binding utility.Substitutions can range from changing or modifying one or more aminoacid residues to complete redesign of a region as long as thetherapeutic utility is maintained Humanized IL-31 antigen bindingmolecules or IL-31 antagonists of the present invention can be can bemodified post-translationally (e.g., acetylation, and phosphorylation)or can be modified synthetically (e.g., the attachment of a labelinggroup). It is understood that the humanized IL-31 antigen bindingmolecules or IL-31 antagonists designed by the present method may haveadditional conservative amino acid substitutions which havesubstantially no effect on antigen binding or other immunoglobulinfunctions.

The humanized IL-31 antigen binding molecules or IL-31 antagonists ofthe present invention include derivatives that are modified, forexample, but not by way of limitation, the derivatives include humanizedIL-31 antigen binding molecules or IL-31 antagonists that have beenmodified, e.g., by glycosylation, acetylation, pegylation,phosphylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

Humanized IL-31 antigen binding molecules or IL-31 antagonists compriseCDRs of a mouse donor immunoglobulin and heavy chain and light chainframeworks of a human acceptor immunoglobulin. Methods of makinghumanized antibody are disclosed in U.S. Pat. Nos. 5,301,101; 5,585,089;5,693,762; and 6,180,370 (each of which is incorporated by reference inits entirety). The CDRs of these antibodies can then be grafted to anyselected human frameworks, which are known in the art, to generate thedesired humanized antibody.

The invention also provides humanized IL-31 antigen binding molecules orIL-31 antagonists that competitively inhibit the binding of a monoclonalantibody described herein to human IL-31. Competitive inhibition can bedetermined by any method known in the art, for example, using thecompetitive binding assays described herein. In preferred embodiments,the antibody competitively inhibits the binding of a monoclonal antibodyof the invention by at least 90%, at least 80%, at least 70%, at least60%, or at least 50% to the polypeptide.

The invention also provides humanized IL-31 antigen binding molecules orIL-31 antagonists that competitively inhibit binding of an antibody toan epitope of the invention as determined by any method known in the artfor determining competitive binding, for example, the immunoassaysdescribed herein. In preferred embodiments, the antibody competitivelyinhibits binding to the epitope by at least 90%, at least 80%, at least70%, at least 60%, or at least 50%.

The in vivo half-lives of the humanized IL-31 antigen binding moleculesor IL-31 antagonists can be increased by modifying (e.g., substituting,deleting or adding) amino acid residues identified as involved in theinteraction between the Fc domain and the FcRn receptor (see, e.g.,International Publication Nos. WO 97/34631 and WO 02/060919, which areincorporated herein by reference in their entireties), or by attachingpolymer molecules such as high molecular weight polyethyleneglycol(PEG). PEG can be attached with or without a multifunctional linkereither through site-specific conjugation of the PEG to the N- orC-terminus of said antibodies or antibody fragments or via epsilon-aminogroups present on lysine residues. Linear or branched polymerderivatization that results in minimal loss of biological activity willbe used. The degree of conjugation will be closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of PEGmolecules to the antibodies. Unreacted PEG can be separated fromantibody-PEG conjugates by, e.g., size exclusion or ion-exchangechromatography.

The present invention includes criteria by which a limited number ofamino acids in the framework of a humanized immunoglobulin chain arechosen to be the same as the amino acids at those positions in the donorrather than in the acceptor, in order to increase the affinity of anantibody comprising the humanized immunoglobulin chain.

In addition to the humanized immunoglobulins specifically describedherein, other “substantially homologous” modified immunoglobulins to thenative sequences can be readily designed and manufactured utilizingvarious recombinant DNA techniques well known to those skilled in theart. A variety of different human framework regions may be used singlyor in combination as a basis for the humanized immunoglobulins of thepresent invention. In general, modifications of the genes may be readilyaccomplished by a variety of well-known techniques, such assite-directed mutagenesis (see, Gillman and Smith, Gene, 8, 81-97 (1979)and S. Roberts et al., Nature, 328, 731-734 (1987), both of which areincorporated herein by reference).

The humanized antibodies of the invention include fragments as well asintact antibodies. Typically, these fragments compete with the intactantibody from which they were derived for antigen binding. The fragmentstypically bind with an affinity of at least 10⁷ M.⁻¹, and more typically10⁸ or 10⁹ M.⁻¹ (ie., within the same ranges as the intact antibody).Humanized antibody fragments include separate heavy chains, light chainsFab, Fab′ F(ab′)₂, and Fv. Fragments are produced by recombinant DNAtechniques, or by enzymic or chemical separation of intactimmunoglobulins.

For details in humanizing antibodies, see European Patent Nos. EP239,400, EP 592,106, and EP 519,596; International Publication Nos. WO91/09967 and WO 93/17105; U.S. Pat. Nos. 5,225,539, 5,530,101,5,565,332, 5,585,089, 5,766,886, and 6,407,213; and Padlan, 1991,Molecular Immunology 28(4/5): 489 498; Studnicka et al., 1994, ProteinEngineering 7(6): 805 814; Roguska et al., 1994, PNAS 91: 969 973; Tanet al., 2002, J. Immunol. 169: 1119 25; Caldas et al., 2000, ProteinEng. 13: 353 60; Morea et al., 2000, Methods 20: 267 79; Baca et al.,1997, J. Biol. Chem. 272: 10678 84; Roguska et al., 1996, Protein Eng.9: 895 904; Couto et al., 1995, Cancer Res. 55 (23 Supp): 5973s 5977s;Couto et al., 1995, Cancer Res. 55: 1717 22; Sandhu, 1994, Gene 150: 40910; Pedersen et al., 1994, J. Mol. Biol. 235: 959 73; Jones et al.,1986, Nature 321: 522-525; Reichmann et al., 1988, Nature 332: 323-329;and Presta, 1992, Curr. Op. Struct. Biol. 2: 593-596.

Various techniques have been developed for the production of antibodyfragments. These fragments can be derived via proteolytic digestion ofintact antibodies (see, e.g., Morimoto et al., Journal of Biochemicaland Biophysical Methods 24: 107-117 (1992) and Brennan et al., Science,229:81 (1985)), or produced directly by recombinant host cells. Forexample, the antibody fragments can be isolated from the antibody phagelibraries discussed above. Alternatively, Fab′-SH fragments can bedirectly recovered from E. coli and chemically coupled to form F(ab′)₂fragments (Carter et al., Bio/Technology 10: 163-167 (1992)). Accordingto another approach, F(ab′) 2 fragments can be isolated directly fromrecombinant host cell culture. Other techniques for the production ofantibody fragments will be apparent to the skilled practitioner.Further, examples of techniques which can be used to producesingle-chain Fvs and antibodies include those described in U.S. Pat.Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra etal., Science 240:1038-1040 (1988).

The present invention encompasses antibodies recombinantly fused orchemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide (or portion thereof, preferably at least10, 20 or 50 amino acids of the polypeptide) of the present invention togenerate fusion proteins. Thus, the invention also pertains toimmunoconjugates comprising the antibody described herein conjugated toa cytotoxic agent such as a chemotherapeutic agent, toxin (e.g. anenzymatically active toxin of bacterial, fungal, plant or animal origin,or fragments thereof), or a radioactive isotope (i.e., aradioconjugate).

The present invention further includes compositions comprising thepolypeptides of the present invention (e.g., those comprising animmunogenic or antigenic epitope) fused or conjugated to heterologouspolypeptide sequences (e.g., antibody domains other than the variableregions). For example, the polypeptides of the present invention may befused or conjugated to an antibody Fc region, or portion thereof. Forexample, polypeptides of the present invention (including fragments orvariants thereof), may be fused with the constant domain ofimmunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (C_(H1),C_(H2), C_(H3), or any combination thereof and portions thereof,resulting in chimeric polypeptides. The antibody portion fused to apolypeptide of the present invention may comprise the constant region,hinge region, C_(H1) domain, C_(H2) domain, and C_(H3) domain or anycombination of whole domains or portions thereof. The polypeptides mayalso be fused or conjugated to the above antibody portions to formmultimers. For example, Fc portions fused to the polypeptides of thepresent invention can form dimers through disulfide bonding between theFc portions. Higher multimeric forms can be made by fusing thepolypeptides to portions of IgA and IgM. Methods for fusing orconjugating the polypeptides of the present invention to antibodyportions are known in the art. See, e.g., U.S. Pat. Nos. 5,336,603;5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 307,434; EP367,166; PCT publications WO 96/04388; WO 91/06570; Ashkenazi et al.,Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J.Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci.USA 89:11337-11341 (1992) (said references incorporated by reference intheir entireties). By way of another non-limiting example, polypeptidesand/or antibodies of the present invention (including fragments orvariants thereof) may be fused with albumin (including but not limitedto recombinant human serum albumin or fragments or variants thereof(see, e.g., U.S. Pat. No. 5,876,969, issued Mar. 2, 1999, EP Patent 0413 622, and U.S. Pat. No. 5,766,883, issued Jun. 16, 1998, hereinincorporated by reference in their entirety)). Polypeptides and/orantibodies of the present invention (including fragments or variantsthereof) may be fused to either the N- or C-terminal end of theheterologous protein (e.g., immunoglobulin Fc polypeptide or human serumalbumin polypeptide). Polynucleotides encoding fusion proteins of theinvention are also encompassed by the invention.

As discussed above, the polypeptides of the present invention may befused or conjugated to the above antibody portions to increase the invivo half life of the polypeptides or for use in immunoassays usingmethods known in the art. Further, the polypeptides of the presentinvention may be fused or conjugated to the above antibody portions tofacilitate purification. One reported example describes chimericproteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. (See, e.g., EP 394,827;Traunecker et al., Nature 331:84-86 (1988)). Enhanced delivery of anantigen across the epithelial barrier to the immune system has beendemonstrated for antigens (e.g., insulin) conjugated to an FcRn bindingpartner such as IgG or Fc fragments (see, e.g., PCT Publications WO96/22024 and WO 99/04813). The polypeptides of the present inventionfused or conjugated to an antibody having disulfide-linked dimericstructures (due to the IgG) may also be more efficient in binding andneutralizing other molecules, than the monomeric secreted protein orprotein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964(1995)). In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinetic properties. (EP A 232,262). Alternatively, deleting theFc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fc portion may hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. (See,D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johansonet al., J. Biol. Chem. 270:9459-9471 (1995)0. Such techniques alsoinclude, but are not limited to, the use of bifunctional conjugatingagents (see e.g., U.S. Pat. Nos. 5,756,065; 5,714,631; 5,696,239;5,652,361; 5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604;5,274,119; 4,994,560; and 5,808,003; the contents of each of which arehereby incorporated by reference in its entirety).

Moreover, the polypeptides of the invention (e.g., antibodies orfragments thereof) can be fused to marker sequences, such as a peptideto facilitates their purification. In a further embodiment, nucleicacids encoding the polypeptides of the invention (including, but notlimited to nucleic acids encoding immunogenic and/or antigenic epitopes)can also be recombined with a gene of interest as an epitope tag (e.g.,the hemagglutinin tag (“HA”) or flag tag) to aid in detection andpurification of the expressed polypeptide. In preferred embodiments, themarker amino acid sequence is a hexa-histidine peptide, such as the tagprovided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth,Calif., 91311), among others, many of which are commercially available.As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824(1989), for instance, hexa-histidine provides for convenientpurification of the fusion protein. Other peptide tags useful forpurification include, but are not limited to, the “HA” tag, whichcorresponds to an epitope derived from the influenza hemagglutininprotein (Wilson et al., Cell 37:767 (1984)) and the “flag” tag.

The present invention further encompasses humanized IL-31 antigenbinding molecules or IL-31 antagonists conjugated to a diagnostic ortherapeutic agent. The IL-31 antigen binding molecules or IL-31antagonists can be used diagnostically to, for example, monitor thedevelopment or progression of a pruritic disease as part of a clinicaltesting procedure to, e.g., determine the efficacy of a given treatment,diagnosis, detection, and/or prevention regimen. Detection can befacilitated by coupling the antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. See,for example, U.S. Pat. No. 4,741,900 for metal ions which can beconjugated to antibodies for use as diagnostics according to the presentinvention. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;examples of suitable prosthetic group complexes includestreptavidin/biotin and avidin/biotin; examples of suitable fluorescentmaterials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ¹¹¹In or ⁹⁹Tc.

Further, humanized IL-31 antigen binding molecules or IL-31 antagonistsmay be conjugated to a therapeutic moiety such as a cytotoxin, e.g., acytostatic or cytocidal agent, a therapeutic agent or a radioactivemetal ion. A cytotoxin or cytotoxic agent includes any agent that isdetrimental to cells. Examples include paclitaxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, a-interferon,.beta.-interferon, nerve growth factor, platelet derived growth factor,tissue plasminogen activator, a thrombotic agent or an anti-angiogenicagent, e.g., angiostatin or endostatin; or, biological responsemodifiers such as, for example, lymphokines, interleukin-1 (“IL-1”),interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophasecolony stimulating factor (“GM-CSF”), granulocyte colony stimulatingfactor (“G-CSF”), or other growth factors.

Humanized IL-31 antigen binding molecules or IL-31 antagonists may alsobe attached to solid supports, which are particularly useful forimmunoassays or purification of the target antigen. Such solid supportsinclude, but are not limited to, glass, cellulose, polyacrylamide,nylon, polystyrene, polyvinyl chloride or polypropylene.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119-58 (1982).

Alternatively, humanized IL-31 antigen binding molecules or IL-31antagonists can be conjugated to a second antibody to form an antibodyheteroconjugate as described by Segal in U.S. Pat. No. 4,676,980.

An humanized IL-31 antigen binding molecules or IL-31 antagonists, withor without a therapeutic moiety conjugated to it, administered alone orin combination with cytotoxic factor(s) and/or cytokine(s) can be usedas a therapeutic.

Humanized IL-31 antigen binding molecules or IL-31 antagonists may beused for tagging cells that express IL-31; for isolating IL-31 byaffinity purification; for diagnostic assays for determining circulatinglevels of IL-31 polypeptides; for detecting or quantitating solubleIL-31 as a marker of underlying pathology or disease; in analyticalmethods employing FACS; for screening expression libraries; forgenerating anti-idiotypic antibodies; and as neutralizing antibodies oras antagonists to block IL-31 activity in vitro and in vivo. Suitabledirect tags or labels include radionuclides, enzymes, substrates,cofactors, inhibitors, fluorescent markers, chemiluminescent markers,magnetic particles and the like; indirect tags or labels may feature useof biotin-avidin or other complement/anti-complement pairs asintermediates. Antibodies herein may also be directly or indirectlyconjugated to drugs, toxins, radionuclides and the like, and theseconjugates used for in vivo diagnostic or therapeutic applications.Moreover, antibodies to IL-31 or fragments thereof may be used in vitroto detect denatured IL-31 or fragments thereof in assays, for example,Western Blots or other assays known in the art.

Suitable detectable molecules may be directly or indirectly attached tothe polypeptide or antibody, and include radionuclides, enzymes,substrates, cofactors, inhibitors, fluorescent markers, chemiluminescentmarkers, magnetic particles and the like. Suitable cytotoxic moleculesmay be directly or indirectly attached to the polypeptide or antibody,and include bacterial or plant toxins (for instance, diphtheria, toxin,saporin, Pseudomonas exotoxin, ricin, abrin and the like), as well astherapeutic radionuclides, such as iodine-131, rhenium-188 or yttrium-90(either directly attached to the polypeptide or antibody, or indirectlyattached through means of a chelating moiety, for instance).Polypeptides or antibodies may also be conjugated to cytotoxic drugs,such as adriamycin. For indirect attachment of a detectable or cytotoxicmolecule, the detectable or cytotoxic molecule can be conjugated with amember of a complementary/anticomplementary pair, where the other memberis bound to the polypeptide or antibody portion. For these purposes,biotin/streptavidin is an exemplary complementary/anticomplementarypair.

The IL-31 antigen binding molecules or IL-31 antagonists of the presentinvention can be measured for their ability to inhibit, block, orneutralize the IL-31 ligand as determined by various in vivo modelsknown in the art and described herein, including but not limited to theNC/Nga model, the Ova epicutaneous model, the chronic hypersensitivitymodel, and the chronic hapten model.

Both skin-homing T cells and epidermal keratinocytes have beenimplicated in the pathology of skin diseases in humans. IL-31 mRNA andprotein expression is restricted to the skin-homing CLA+ T cellpopulation in humans. See U.S. patent application Ser. No. 11/353,427,filed Feb. 14, 2006, (U.S. Patent Publication No. 2006-0188499) and U.S.patent application Ser. No. 11/353,454, filed Feb. 14, 2006, (U.S.Patent Publication No. 2006-0188500), both of which are incorporatedherein by reference. As such, an antagonist to IL-31, including anantibody or receptor antagonist will be useful in treating skin andepidermal diseases which have expression of CLA+ T cells. Such diseasesinclude, for example, atopic dermatitis, contact dermatitis,drug-induced allergic reactions, skin-tropic viruses and viralassociated pruritis, vitiligo, cutaneous T cell lymphoma, alopeciaaerata, acne rosacea, acne vulgaris, prurigo nodularis, and bullouspemphigoid. Chemokine markers such as TARC and MDC are useful to measurethe effect of a neutralizing monoclonal antibody to IL-31. Theinhibitory effects of treatment with humanized IL-31 antigen bindingmolecules or IL-31 antagonists described herein can be measured bymonitoring the levels of TARC and MDC.

Contact Dermatitis

Allergic contact dermatitis is defined as a T cell mediated immunereaction to an antigen that comes into contact with the skin. The CLA+ Tcell population is considered to be involved in the initiation ofdermatitis since allergen dependent T cell responses are largelyconfined to the CLA+ population of cells (See Santamaria-Babi, L. F., etal., J Exp Med: 181, 1935, (1995)). Recent data has found that onlymemory (CD45RO+) CD4+ CLA+ and not CD8+ T cells proliferate and produceboth type-1 (IFN-) and type-2 (IL-5) cytokines in response to nickel, acommon contact hypersensitivity allergen. Furthermore, cells expressingCLA in combination with CD4, CD45RO (memory) or CD69 are increased afternickel-specific stimulation and express the chemokine receptors CXCR3,CCR4, CCR10 but not CCR6. See Moed H., et al., Br J Dermatol: 51, 32,(2004).

In animal models, it has been demonstrated that allergic contactdermatitis is T-cell dependent and that the allergic-responsive T cellsmigrate to the site of allergen application. See generally: Engeman TM., et al., J Immunol: 164, 5207, (2000); Ferguson T. A. & Kupper T. S.J Immunol: 150, 1172, (1993); and Gorbachev A. V. & Fairchild R. L. CritRev Immunol: 21, 451(2001). Since CLA+ T cells produce IL-31 and IL-31stimulation of skin keratinocytes can induce pro-inflammatorychemokines, such as TARC and MDC, IL-31 may be involved in thepathophysiology of contact dermatitis. By using a neutralizing IL-31antibody in a mouse model of contact hypersensitivity.

Thus, neutralization of IL-31 by the humanized IL-31 antigen bindingmolecules or IL-31 antagonists described herein may be used to improveclinical outcome of Contat Hypersenstivity by inhibition, reduction,neutralization, prevention or blocking the inflammation and/orscratching associated with disease.

Atopic Dermatitis

Atopic dermatitis (AD) is a chronically relapsing inflammatory skindisease with a dramatically increasing incidence over the last decades.Clinically AD is characterized by highly pruritic often excoriatedplaques and papules that show a chronic relapsing course. The diagnosisof AD is mostly based on major and minor clinical findings. See HanifinJ M, Arch Dermatol: 135, 1551 (1999). Histopathology reveals spongiosis,hyper and focal parakeratosis in acute lesions, whereas marked epidermalhyperplasia with hyper and parakeratosis, acanthosis/hypergranulosis andperivascular infiltration of the dermis with lymphocytes and abundantmast cells are the hallmarks of chromic lesions.

T cells play a central role in the initiation of local immune responsesin tissues and evidence suggests that skin-infiltrating T cells inparticular, may play a key role in the initiation and maintenance ofdisregulated immune responses in the skin. Approximately 90% ofinfiltrating T cells in cutaneous inflammatory sites express thecutaneous lymphocyte-associated Ag (CLA+) which binds E-selectin, aninducible adhesion molecule on endothelium (reviewed in Santamaria-BabiL. F., et al., Eur J Dermatol: 14, 13, (2004)). A significant increasein circulating CLA+ T cells among AD patients compared with controlindividuals has been documented (See Teraki Y., et al., Br J Dermatol:143, 373 (2000), while others have demonstrated that memory CLA+ T cellsfrom AD patients preferentially respond to allergen extract compared tothe CLA-population (See Santamaria-Babi, L. F., et al., J Exp Med: 181,1935, (1995)). In humans, the pathogenesis of atopic disorders of theskin have been associated with increases in CLA+ T cells that expressincreased levels of Th-2-type cytokines like IL-5 and IL-13 9, 10. SeeAkdis M., et al., Eur J Immunol: 30, 3533 (2000); and Hamid Q., et al.,J Allergy Clin Immunol: 98, 225 (1996).

NC/Nga Mice spontaneously develop AD-like lesions that parallel human ADin many aspects, including clinical course and signs, histophathologyand immunopathology when housed in non-specified pathogen-free (non-SPF)conditions at around 6-8 weeks of age. In contrast, NC/Nga mice keptunder SPF conditions do not develop skin lesions. However, onset ofspontaneous skin lesions and scratching behaviour can be synchronized inNC/Nga mice housed in a SPF facility by weekly intradermal injection ofcrude dust mite antigen. See Matsuoka H., et al., Allergy: 58, 139(2003). Therefore, the development of AD in NC/Nga is a useful model forthe evaluation of novel therapeutics for the treatment of AD.

In addition to the NC/Nga model of spontaneous AD, epicutaneoussensitization of mice using OVA can also be used as a model to induceantigen-dependent epidermal and dermal thickening with a mononuclearinfiltrate in skin of sensitized mice. This usually coincides withelevated serum levels of total and specific IgE, however no skin barrierdysfunction or pruritus normally occurs in this model. See Spergel J.M., et al., J Clin Invest, 101: 1614, (1998). This protocol can bemodified in order to induce skin barrier disregulation and pruritis bysensitizing D011.10 OVA TCR transgenic mice with OVA. Increasing thenumber of antigen-specific T cells that could recognize the sensitizingantigen may increase the level of inflammation in the skin to inducevisible scratching behaviour and lichenification/scaling of the skin.

Both the NC/Nga spontaneous AD model and the OVA epicutaneous D011.10model are used to evaluate the ability of the humanized IL-31 antigenbinding molecules or IL-31 antagonists described herein to inhibit,reduce, or neutralize the effects of IL-31. Administration of humanizedIL-31 antigen binding molecules or IL-31 antagonists can result in areduction in scratching that can be effective in treating pruriticdiseases including, but not limited to, atopic dermatitis, prurigonodularis, and eczema, since cessation of scratching will stopprogression of dermatitis, the development of which is dependent onscratching.

Additional models to measure the inhibitory effects of the humanizedIL-31 antigen binding molecules or IL-31 antagonists described hereinare described by Umeuchi, H. et al., European Journal of Pharmacology,518: 133-139, 2005; and by Yoo, J. et al., J. Experimental Medicine,202:541-549, 2005.

Thus, neutralization of IL-31 by the humanized IL-31 antigen bindingmolecules or IL-31 antagonists described herein may be used to improveclinical outcome of dermatitis and pruritic diseases including atopicdermatitis, prurigo nodularis, and eczema by inhibition, reduction,prevention or blocking the inflammation and/or scratching associatedwith disease.

Methods of measuring the ability of the humanized anti-IL-31 antibodiesand antagonists to inhibit, reduce, or neutralize the itch responseinclude the following assays and models:

I) Capsaicin Treatment of IL-31 Treated Mice

Ten week old BALB/c animals (CRL) are anaesthetized and injected with along-lasting analgesic agent, bupranorphine hydrochloride,subcutaneously at 0.1 mg/kg before injection of 0.25 ml of 4 mg/mlsolution of capsaicin in 10% ethanol+10% Tween-80 in salinesubcutaneously into scruff of neck. Animals are kept anaesthetized forat least 30 min following neurotoxin treatment. Forty-eight hours later,14-day osmotic pumps are implanted subcutaneously for continuousdelivery of 20 ug/day of IL-31 for 14 days. Mice are monitored daily for6 days for alopecia and pruritis using the following criteria: 0=noscratching, animal appears normal, 1=thinning of coat in small areas,scratching noted, 2=minor hair loss (small patches), scratching,3=moderate hair loss, scratching, and 4=severe hair loss, excessivescratching.

Neutralization, inhibition, or reduction of IL-31 by humanized IL-31antigen binding molecules or IL-31 antagonists may decrease theincidence and intensity of itch, and therefore dermatitis, in patientssuffering from skin disorders that involve itch.

II) Tac1 Gene Expression

Mice that are homozygous null for the Tac1 gene express no detectablesubstance P or neurokinin A. These mice have significantly reducednociceptive pain responses to moderate to intense stimuli and aretherefore a useful tool for studying the contribution of tachykininpeptides to pain/itch processing and inflammatory disease states. Twelveweek old, Tac1 knockout mice were implanted with 14-day osmotic pumpsdelivering 1 ug/day of IL-31 protein and observed daily for alopecia andpruritis using the following criteria: 0=no scratching, animal appearsnormal, 1=thinning of coat in small areas, scratching noted, 2=minorhair loss (small patches), scratching, 3=moderate hair loss, scratching,and 4=severe hair loss, excessive scratching.

Results of this study show that Tact deficient mice were lesssusceptible to IL-31 induced scratching/hairloss compared to wildtypecontrol mice. While 100% (10/10) of wildtype mice had developed evidenceof scratching and hairloss by day 6 of IL-31 treatment, only 33.3% (2/6)Tac1 deficient mice were showing signs of scratching and hairloss at thesame time-point. Thus, neutralization, inhibition or reduction of IL-31by humanized IL-31 antigen binding molecules or IL-31 antagonists maydecrease the incidence and intensity of scratching in the context ofdermatitis.

III) Administration of IL-31 Neutralizing Antibody

Normal female BALB/c mice (CRL) approximately 8 to 12 weeks old can beimplanted subcutaneously with 14-day osmotic pumps (Alzet, #2002)delivering 1 ug/day mIL-31. Groups of mice receive intraperitoneal(i.p.) injections of rat anti-mouse IL-31 monoclonal antibody 10 mg/kg(200 ug/mouse) twice weekly starting 1 week prior to IL-31 delivery.Control groups of mice receive i.p. injections of vehicle (PBS/0.1% BSA)with the identical dosing schedules. Mice are scored daily for alopeciaand pruritis using the following criteria: 0=no scratching, animalappears normal, 1=thinning of coat in small areas, scratching noted,2=minor hair loss (small patches), scratching, 3=moderate hair loss,scratching, and 4=severe hair loss, excessive scratching.

Thus, neutralization, reduction or inhibition of IL-31 by IL-31 antigenbinding molecules or IL-31 antagonists may delay the onset of thescratch/hairloss response induced by IL-31.

The effects of humanized IL-31 antigen binding molecules or IL-31antagonists are measured by inhibition of scratching, itching,dermatitis, a reduction in IL-31RA expression in kerotinocytes, and/or areduction in score for alopecia and pruritis.

Drug-Induced Delayed Type Cutaneous Allergic Reactions

Drug-induced delayed type cutaneous allergic reactions are veryheterogeneous and may mirror many distinct pathophysiological events.See Brockow K., et al., Allergy: 57, 45 (2002). Immunological mechanismsinvolved in these reactions have been shown as either antibody or cellmediated. In immediate drug allergy an IgE-mediated antibody reactioncan be demonstrated by a positive skin prick and/or intradermal testafter 20 min, whereas non-immediate reactions to drugs can occur morethan one hour after last drug intake and are often T-cell mediated.Non-immediate T-cell mediated delayed type reactions can occur inpatients with adverse drug reactions to penicillins for exampleProliferative T cell responses to penicillins have been shown to berestricted to the memory (CD45RO+) CLA+ subpopulation of T cells frompenicillin allergic patients whereas the CD45RO+CLA− subset shows noproliferative response. See Blanca M., Leyva L., et al., Blood Cells MolDis: 31, 75 (2003). Delayed-type hypersensitivity (DTH) reactions can beartificially reproduced in mice, allowing assessment of factors that maybe involved in the initiation and perpetuation of the DTH response.Humanized IL-31 antigen binding molecules or IL-31 antagonists could beeffective in limiting, reducing, inhibiting a delayed typehypersensitivity reaction.

Toxic epidermal necrolysis (TEN) is a very rare but extremely severedrug reaction characterized by widespread apoptosis of epidermis withextensive blisters. Studies have shown that lymphocytes infiltrating theblister are CLA+ T cells and can exhibit cytotoxicity towards epidermalkeratinocytes. See Leyva L., et al., J Allergy Clin Immunol: 105, 157(2000); and Nassif A., Bensussan A., et al., J Allergy Clin Immunol:114, 1209 2004). A transgenic mouse system, whereby OVA is expressedunder the control of the keratin-5 (K5) promoter in the epidermal andhair follicular keratinocytes of mice, has been generated to establishan animal model for TEN. OVA specific CD8+ T cells, when adoptivelytransferred into K5-OVA mice, undergo activation and proliferation inthe skin-draining lymph nodes and target the skin of K5-OVA mice,resulting in development of skin lesions that are reminiscent of TEN.See Azukizawa H., et al., Eur J Immunol: 33, 1879 (2003).

Thus, neutralization of IL-31 by the humanized IL-31 antigen bindingmolecules or IL-31 antagonists described herein may be used to improveclinical outcome of TEN by inhibition, reduction, prevention or blockingthe inflammation and/or scratching associated with disease.

Bullous Pemphigoid

Bullous pemphigoid is a subepidermal disorder which manifests assubepidermal blisters with a dermal infiltrate of neutrophils andeosinophils. Diagnosis is characterized by the presence ofantigen-specific antibodies against specific adhesion proteins of theepidermis and dermal-epidermal junction. See Jordon R. E., et al., 0:200, 751 (1967). Studies analyzing the role of T cells in thepathogenesis of bullous pemphigoid by analysis of PBL and skin blister Tcells have found a predominance of CLA+ T cells expressing increasedlevels of Th2-cytokines like IL-4 and IL-13. See Teraki Y., et al., JInvest Dermatol: 117, 1097 (2001). In bullous pemphigoid patientsfollowing therapy with systemic corticosteroids, the frequency of CLA+,but not CLA−, interleukin-13-producing cells is significantly decreased.Decreases in CLA+ cells following corticosteroid treatment is associatedwith clinical improvement. See Teraki, ibid.

Thus, neutralization of IL-31 by the humanized IL-31 antigen bindingmolecules or IL-31 antagonists described herein may be used to improveclinical outcome of bullous pemphigoid by inhibition, reduction,prevention or blocking the inflammation and/or scratching associatedwith disease.

Alopecia Areata

Alopecia areata (AA) is regarded as a tissue-restricted autoimmunedisease of hair follicles in which follicular activity is arrestedbecause of the continued activity of lymphocytic infiltrates. AA resultsin patches of complete hair loss anywhere on the body, though actualloss of hair follicles does not occur, even in hairless lesions.Although clinical signs of inflammation are absent, skin biopsies fromsites of active disease show perifollicular lymphocytic inflammation ofprimarily CD4+ cells, along with a CD8+ intrafollicular infiltrate. SeeKalish R. S. & Gilhar A. J Investig Dermatol Symp Proc: 8, 164 (2003).

Studies have shown that scalp skin infiltrating CD4+ or CD8+ lymphocytesexpress CLA and, in peripheral blood of individuals with AA, the percentof CLA+ CD4+ or CD8+ lymphocytes is significantly higher than that ofnormal controls. Furthermore, patients with severe or progressive AAshow a much higher CLA-positivity compared to patients recovering fromthe disease and a decrease in percent CLA+ cells parallels a goodclinical course. See Yano S., et al., Acta Derm Venereol: 82, 82 (2002).These studies therefore suggest that CLA+ lymphocytes may play animportant role in AA. Xenograft models have demonstrated that activatedT cells are likely to play a role in the pathogenesis of AA. Lesionalscalp from AA patients grafted onto nude mice regrows hair coincidentwith a loss of infiltrating lymphocytes from the graft and, transfer ofactivated lesional T cells to SCID mice can transfer hair loss to humanscalp explants on SCID mice. See Kalish R. S. & Gilhar A. J InvestigDermatol Symp Proc: 8, 164 (2003).

A variety of immunomodulating therapies are part of the usual treatmentfor this disorder however none of these treatments have been consistentin their efficacy. See Tang L., et al., J Invest Dermatol: 120, 400(2003); Tang L., et al., (2004); and Tang L., et al., J Am AcadDermatol: 49, 1013 (2003). Nevertheless, their uses in valid animalmodels provide a tool to dissect out molecular mechanisms of therapeuticeffects. See Shapiro J., et al., J Investig Dermatol Symp Proc: 4, 239(1999); Tang L., et al., Old wine in new bottles: reviving old therapiesfor alopecia areata using rodent models (2003); and Verma D. D., et al.,Eur J Dermatol: 14, 332 (2004).

Thus, neutralization of IL-31 by the humanized IL-31 antigen bindingmolecules or IL-31 antagonists described herein may be used to improveclinical outcome of alopecia areata by inhibition, reduction, preventionor blocking the inflammation and/or scratching associated with disease.

Acne Rosacea/Acne Vulgaris

Acne vulgaris, a disorder of the pilosebaceous apparatus, is the mostcommon skin problem of adolescence. Abnormalities in follicularkeratinization are thought to produce the acne lesion. Acne rosacea isdifferentiated from acne vulagaris by the presence of red papules,pustules, cysts and extensive telangiectasias, but the absence ofcomedones (white heads). Increased sebum excretion from sebaceous glandsis a major factor in the pathophysiology of acne vulgaris. Othersebaceous gland functions are also associated with the development ofacne, including sebaceous proinflammatory lipids; different cytokinesproduced locally; periglandular peptides and neuropeptides, such ascorticotrophin-releasing hormone, which is produced by sebocytes; andsubstance P, which is expressed in the nerve endings at the vicinity ofhealthy-looking glands of acne patients. See Zouboulis C. C. ClinDermatol: 22, 360 (2004).

Although the pathophysiology of acne vulgaris and acne rosacea remainsunknown, clinical observations and histopathologic studies suggest thatinflammation of the pilosebaceous follicle may be central to thepathogenesis of rosacea and acne vulgaris. Early studies on analysis ofT cell subsets infiltrating rosacea legions indicated that the majorityof T cells expressed CD4. See Rufli T. & Buchner S. A. Dermatologica:169, 1 (1984).

CD4+ T cells produce IL-31 and IHC analysis of skin for IL-31 expressionsuggests that IL-31 is expressed in sebaceous and sweat glands. IL-31stimulation of epidermal keratinocytes induces expression of chemokineswhich likely results in cellular infiltration suggesting that IL-31 maycontribute to the pro-inflammatory response in skin. See Dillon S. R.,et al., Nat Immunol: 5, 752 (2004). IL-31 may therefore contribute tothe pathophysiology of acne rosacea and acne vulgaris.

Thus, neutralization of IL-31 by the humanized IL-31 antigen bindingmolecules or IL-31 antagonists described herein may be used to improveclinical outcome of acne vulgaris by inhibition, reduction, preventionor blocking the inflammation and/or scratching associated with disease.

Prurigo Nodularis

Prurigo nodularis is an eruption of lichenified or excoriated nodulescaused by intractable pruritus that is difficult to treat. While chronicrubbing results in lichenification, and scratching in linearexcoriations, individuals who pick and gouge at their itchy, irritatedskin tend to produce markedly thickened papules known as prurigonodules. Although prurigo nodularis is not specific to atopicdermatitis, many patients with these nodules also have an atopicreaction, which manifests as allergic rhinitis, asthma, or food allergy.T cells represent the majority of infiltrating cells in prurigo lesionsand these lesions often represents the most pruritic skin lesion inatopy patients.

Topical treatment of prurigo nodularis with capsaicin, an anti-pruriticalkaloid that interferes with the perception of prurities and pain bydepletion of neuropeptides like substance P in small sensory cutaneousnerves, has proven to be an effective and safe regimen resulting inclearing of the skin lesions. See Stander S., et al., J Am AcadDermatol: 44, 471 (2001). Studies of the itch response in NC/Nga miceusing capsaicin treatment showed that the spontaneous development ofdermatitis lesions was almost completely prevented. Furthermore, theelevation of serum IgE levels was significantly suppressed andinfiltrating eosinophils and mast cell numbers in lesional skin ofcapsaicin treated mice were reduced. See Mihara K., et al., Br JDermatol: 151, 335 (2004). The observations from this group suggest thatscratching behaviour might contribute to the development of dermatitisby enhancing various immunological responses, therefore implying thatprevention of the itch sensation and/or itch-associated scratchingbehaviour might be an effective treatment for AD. See Mihara K., et al.,Br J Dermatol: 151, 335 (2004). Thus, the humanized anti-IL-31antibodies described herein will be useful in minimizing the effects ofAD, prurigo nodularis, and other pruritic diseases as they are shownherein to reduce the amount of scratching in NC/Nga mice.

Chronic delivery of IL-31 induces pruritis and alopecia in mice followedby the development of skin lesions resembling dermatitis suggesting thatIL-31 may induce itching. See Dillon S. R., et al., Nat Immunol: 5, 752(2004). The involvement of IL-31 in induction of the itch response canbe measured, for example, by two methods (i) capsaicin treatment ofIL-31-treated mice; and (ii) IL-31 treatment of Tac1 knockout mice,which have significantly reduced nociceptive pain responses because oflack of expression of neuropeptides. In addition, whether neutralizationof IL-31 in IL-31 treated mice with humanized IL-31 antigen bindingmolecules or IL-31 antagonists could prevent pruritis and alopecia canbe tested in these models.

Thus, neutralization of IL-31 by the humanized IL-31 antigen bindingmolecules or IL-31 antagonists described herein may be used to improveclinical outcome of prurigo nodularis by inhibition, reduction,prevention or blocking the inflammation and/or scratching associatedwith disease.

Skin-Tropic Viruses and Viral Associated Pruritis

Herpes Simplex Virus (HSV)-specific CD8+ T cells in the peripheral bloodand HSV-specific CD8+ T cells recovered from herpes lesions express highlevels of CLA where as non-skin-tropic herpes virus-specific CD8+ Tcells lack CLA expression. See Koelle D. M., et al., J Clin Invest: 110,537 (2002). HSV-2 reactive CD4+ T lymphocytes also express CLA, but atlevels lower than those previously observed for CD8+ T lymphocytes. SeeGonzalez J. C., et al., J Infect Dis: 191, 243 (2005). Pruritis has alsobeen associated with herpes viral infections (See Hung K. Y., et al.,Blood Purif: 16, 147 (1998). though other viral diseases, like HIV, havealso been associated with pruritic skin lesions. Severe, intractablepruritus, often associated with erythematopapular skin lesions andhypereosinophilia, is a condition observed in some nonatopic,HIV-infected patients 36. See Singh F. & Rudikoff D, Am J Clin Dermatol;4, 177 (2003); and Milazzo F., Piconi S., et al., Allergy: 54, 266(1999).

The association of skin-tropic viruses with pruritis and CLA+ T cellssuggests that IL-31 producing T cells may be involved in thepathophysiology of viral infections.

Thus, neutralization of IL-31 by the humanized IL-31 antigen bindingmolecules or IL-31 antagonists described herein may be used to improveclinical outcome of pruritis associated with skin-tropic viruses byinhibition, reduction, prevention or blocking the inflammation and/orscratching associated with disease.

Moreover, inflammation is a protective response by an organism to fendoff an invading agent. Inflammation is a cascading event that involvesmany cellular and humoral mediators. On one hand, suppression ofinflammatory responses can leave a host immunocompromised; however, ifleft unchecked, inflammation can lead to serious complications includingchronic inflammatory diseases (e.g., rheumatoid arthritis, multiplesclerosis, inflammatory bowel disease and the like), septic shock andmultiple organ failure. Importantly, these diverse disease states sharecommon inflammatory mediators. The collective diseases that arecharacterized by inflammation have a large impact on human morbidity andmortality. Therefore it is clear that anti-inflammatory antibodies andbinding polypeptides, such as anti-IL-31 antibodies and bindingpolypeptides described herein, could have crucial therapeutic potentialfor a vast number of human and animal diseases, from asthma and allergyto autoimmunity and septic shock. As such, use of anti-inflammatory antiIL-31 antibodies and binding polypeptides described herein can be usedtherapeutically as IL-31 antagonists described herein, particularly indiseases such as arthritis, endotoxemia, inflammatory bowel disease,psoriasis, related disease and the like.

1. Arthritis

Arthritis, including osteoarthritis, rheumatoid arthritis, arthriticjoints as a result of injury, and the like, are common inflammatoryconditions which would benefit from the therapeutic use ofanti-inflammatory antibodies and binding polypeptides, such asanti-IL-31 antibodies and binding polypeptides of the present invention.For Example, rheumatoid arthritis (RA) is a systemic disease thataffects the entire body and is one of the most common forms ofarthritis. It is characterized by the inflammation of the membranelining the joint, which causes pain, stiffness, warmth, redness andswelling. Inflammatory cells release enzymes that may digest bone andcartilage. As a result of rheumatoid arthritis, the inflamed jointlining, the synovium, can invade and damage bone and cartilage leadingto joint deterioration and severe pain amongst other physiologiceffects. The involved joint can lose its shape and alignment, resultingin pain and loss of movement.

Rheumatoid arthritis (RA) is an immune-mediated disease particularlycharacterized by inflammation and subsequent tissue damage leading tosevere disability and increased mortality. A variety of cytokines areproduced locally in the rheumatoid joints. Numerous studies havedemonstrated that IL-1 and TNF-alpha, two prototypic pro-inflammatorycytokines, play an important role in the mechanisms involved in synovialinflammation and in progressive joint destruction. Indeed, theadministration of TNF-alpha and IL-1 inhibitors in patients with RA hasled to a dramatic improvement of clinical and biological signs ofinflammation and a reduction of radiological signs of bone erosion andcartilage destruction. However, despite these encouraging results, asignificant percentage of patients do not respond to these agents,suggesting that other mediators are also involved in the pathophysiologyof arthritis (Gabay, Expert. Opin. Biol. Ther. 2(2): 135-149, 2002). Oneof those mediators could be IL-31, and as such a molecule that binds orinhibits IL-31, such as anti IL-31 antibodies or binding partners, couldserve as a valuable therapeutic to reduce inflammation in rheumatoidarthritis, and other arthritic diseases.

There are several animal models for rheumatoid arthritis known in theart. For example, in the collagen-induced arthritis (CIA) model, micedevelop chronic inflammatory arthritis that closely resembles humanrheumatoid arthritis. Since CIA shares similar immunological andpathological features with RA, this makes it an ideal model forscreening potential human anti-inflammatory compounds. The CIA model isa well-known model in mice that depends on both an immune response, andan inflammatory response, in order to occur. The immune responsecomprises the interaction of B-cells and CD4+ T-cells in response tocollagen, which is given as antigen, and leads to the production ofanti-collagen antibodies. The inflammatory phase is the result of tissueresponses from mediators of inflammation, as a consequence of some ofthese antibodies cross-reacting to the mouse's native collagen andactivating the complement cascade. An advantage in using the CIA modelis that the basic mechanisms of pathogenesis are known. The relevantT-cell and B-cell epitopes on type II collagen have been identified, andvarious immunological (e.g., delayed-type hypersensitivity andanti-collagen antibody) and inflammatory (e.g., cytokines, chemokines,and matrix-degrading enzymes) parameters relating to immune-mediatedarthritis have been determined, and can thus be used to assess testcompound efficacy in the CIA model (Wooley, Curr. Opin. Rheum. 3:407-20,1999; Williams et al., Immunol. 89:9784-788, 1992; Myers et al., LifeSci. 61:1861-78, 1997; and Wang et al., Immunol. 92:8955-959, 1995).

As a molecule that modulates immune and inflammatory response, IL-31 mayinduce production of SAA, which is implicated in the pathogenesis ofrheumatoid arthritis. Humanized IL-31 antigen binding molecules or IL-31antagonists may reduce SAA activity in vitro and in vivo, the systemicor local administration of IL-31 antigen binding molecules or IL-31antagonists can potentially suppress the inflammatory response in RA.

2. Endotoxemia

Endotoxemia is a severe condition commonly resulting from infectiousagents such as bacteria and other infectious disease agents, sepsis,toxic shock syndrome, or in immunocompromised patients subjected toopportunistic infections, and the like. Therapeutically useful ofanti-inflammatory antibodies and binding polypeptides, such asanti-IL-31 antibodies and binding polypeptides of the present invention,could aid in preventing and treating endotoxemia in humans and animalsOther potential therapeutics include IL-31RA polypeptides, solubleheterodimeric and multimeric receptor polypeptides, or anti IL-31antibodies or binding partners of the present invention, and the like,could serve as a valuable therapeutic to reduce inflammation andpathological effects in endotoxemia.

Lipopolysaccharide (LPS) induced endotoxemia engages many of theproinflammatory mediators that produce pathological effects in theinfectious diseases and LPS induced endotoxemia in rodents is a widelyused and acceptable model for studying the pharmacological effects ofpotential pro-inflammatory or immunomodulating agents. LPS, produced ingram-negative bacteria, is a major causative agent in the pathogenesisof septic shock (Glausner et al., Lancet 338:732, 1991). A shock-likestate can indeed be induced experimentally by a single injection of LPSinto animals Molecules produced by cells responding to LPS can targetpathogens directly or indirectly. Although these biological responsesprotect the host against invading pathogens, they may also cause harm.Thus, massive stimulation of innate immunity, occurring as a result ofsevere Gram-negative bacterial infection, leads to excess production ofcytokines and other molecules, and the development of a fatal syndrome,septic shock syndrome, which is characterized by fever, hypotension,disseminated intravascular coagulation, and multiple organ failure(Dumitru et al. Cell 103:1071-1083, 2000).

These toxic effects of LPS are mostly related to macrophage activationleading to the release of multiple inflammatory mediators. Among thesemediators, TNF appears to play a crucial role, as indicated by theprevention of LPS toxicity by the administration of neutralizinganti-TNF antibodies (Beutler et al., Science 229:869, 1985). It is wellestablished that 1 ug injection of E. coli LPS into a C57Bl/6 mouse willresult in significant increases in circulating IL-6, TNF-alpha, IL-1,and acute phase proteins (for example, SAA) approximately 2 hours postinjection. The toxicity of LPS appears to be mediated by these cytokinesas passive immunization against these mediators can result in decreasedmortality (Beutler et al., Science 229:869, 1985). The potentialimmunointervention strategies for the prevention and/or treatment ofseptic shock include anti-TNF mAb, IL-1 receptor antagonist, LIF, IL-10,and G-CSF. Since LPS induces the production of pro-inflammatory factorspossibly contributing to the pathology of endotoxemia, theneutralization of IL-31 activity, SAA or other pro-inflammatory factorsby antagonizing IL-31 polypeptide can be used to reduce the symptoms ofendotoxemia, such as seen in endotoxic shock. Other potentialtherapeutics include humanized IL-31 antigen binding molecules or IL-31antagonists.

3. Inflammatory Bowel Disease. IBD

Inflammatory Bowel Disease (IBD) can affect either colon and rectum(Ulcerative colitis) or both, small and large intestine (Crohn'sDisease). The pathogenesis of these diseases is unclear, but theyinvolve chronic inflammation of the affected tissues. Potentialtherapeutics include IL-31RA polypeptides, soluble heterodimeric andmultimeric receptor polypeptides, or anti-IL-31 antibodies or bindingpartners of the present invention, and the like, could serve as avaluable therapeutic to reduce inflammation and pathological effects inIBD and related diseases.

The chronic inflammation and ulceration in Crohn's disease usuallystarts with either small-intestinal obstruction or abdominal pain whichmay mimic acute appendicitis; other presentations can relate to itscomplications. The course of the disease is chronic, and there may beexacerbations and remissions in spite of therapy. Onset is usually inearly adult life, with about half of all cases beginning between theages of 20 and 30 years and 90% between 10 and 40 years. Slightly moremales than females are affected.

Microscopy reflects the gross appearances. Inflammation involvement isdiscontinuous: it is focal or patchy. Collections of lymphocytes andplasma cells are found mainly in the mucosa and submucosa but usuallyaffecting all layers (transmural inflammation). The classicalmicroscopic feature of Crohn's disease is the presence of granule cellssurrounded by a cuff of lymphocytes. The incidence of idiopathicinflammatory bowel diseases shows considerable geographic variation.These diseases have a much higher incidence in northern Europe and theUnited States than in countries of southern Europe, Africa, SouthAmerica and Asia, although increasing urbanisation and prosperity isleading to a higher incidence in parts of southern Europe and Japan(General and Systematic Pathology, Churchill Livingstone, 3rd edition2000, JCE Underwood, Ed.).

In Crohn's disease, clinically there are two main groups, the firstcomprising patients whose disease goes into lasting remission withinthree years of onset, the second comprising patients with diseasepersisting beyond three years.

Whatever the aetiology, there is evidence of persistence andinappropriate T-cell and macrophage activation in Crohn's disease withincreased production of pro-inflammatory cytokines, in particularinterleukins (IL) 1, 2, 6 and 8, Interferon (IFN)- and Tumor NecrosisFactor (TNF). Crohn's disease is characterised by sustained (chronic)inflammation accompanied by fibrosis. The process of fibroblasticproliferation and collagen deposition may be mediated by transforminggrowth factor, which has certain anti-inflammatory actions, namelyfibroblast recruitment, matrix synthesis and down-regulation ofinflammatory cells, but it is likely that many other mediators will beimplicated

Ulcerative colitis (UC) is an inflammatory disease of the largeintestine, commonly called the colon, characterized by inflammation andulceration of the mucosa or innermost lining of the colon. Thisinflammation causes the colon to empty frequently, resulting indiarrhea. Symptoms include loosening of the stool and associatedabdominal cramping, fever and weight loss. Although the exact cause ofUC is unknown, recent research suggests that the body's natural defensesare operating against proteins in the body which the body thinks areforeign (an “autoimmune reaction”). Perhaps because they resemblebacterial proteins in the gut, these proteins may either instigate orstimulate the inflammatory process that begins to destroy the lining ofthe colon. As the lining of the colon is destroyed, ulcers formreleasing mucus, pus and blood. The disease usually begins in the rectalarea and may eventually extend through the entire large bowel. Repeatedepisodes of inflammation lead to thickening of the wall of the intestineand rectum with scar tissue. Death of colon tissue or sepsis may occurwith severe disease. The symptoms of ulcerative colitis vary in severityand their onset may be gradual or sudden. Attacks may be provoked bymany factors, including respiratory infections or stress.

Although there is currently no cure for UC available, treatments arefocused on suppressing the abnormal inflammatory process in the colonlining Treatments including corticosteroids immunosuppressives (eg.azathioprine, mercaptopurine, and methotrexate) and aminosalicytates areavailable to treat the disease. However, the long-term use ofimmunosuppressives such as corticosteroids and azathioprine can resultin serious side effects including thinning of bones, cataracts,infection, and liver and bone marrow effects. In the patients in whomcurrent therapies are not successful, surgery is an option. The surgeryinvolves the removal of the entire colon and the rectum.

There are several animal models that can partially mimic chroniculcerative colitis. The most widely used model is the2,4,6-trinitrobenesulfonic acid/ethanol (TNBS) induced colitis model,which induces chronic inflammation and ulceration in the colon. WhenTNBS is introduced into the colon of susceptible mice via intra-rectalinstillation, it induces T-cell mediated immune response in the colonicmucosa, in this case leading to a massive mucosal inflammationcharacterized by the dense infiltration of T-cells and macrophagesthroughout the entire wall of the large bowel. Moreover, thishistopathologic picture is accompanies by the clinical picture ofprogressive weight loss (wasting), bloody diarrhea, rectal prolapse, andlarge bowel wall thickening (Neurath et al. Intern. Rev. Immunol.19:51-62, 2000).

Another colitis model uses dextran sulfate sodium (DSS), which inducesan acute colitis manifested by bloody diarrhea, weight loss, shorteningof the colon and mucosal ulceration with neutrophil infiltration.DSS-induced colitis is characterized histologically by infiltration ofinflammatory cells into the lamina propria, with lymphoid hyperplasia,focal crypt damage, and epithelial ulceration. These changes are thoughtto develop due to a toxic effect of DSS on the epithelium and byphagocytosis of lamina propria cells and production of TNF-alpha andIFN-gamma. Despite its common use, several issues regarding themechanisms of DSS about the relevance to the human disease remainunresolved. DSS is regarded as a T cell-independent model because it isobserved in T cell-deficient animals such as SCID mice.

The administration of humanized IL-31 antigen binding molecules or IL-31antagonists to these TNBS or DSS models can be used to evaluate the useof IL-31 antagonists to ameliorate symptoms and alter the course ofgastrointestinal disease. IL-31 may play a role in the inflammatoryresponse in colitis, and the neutralization of IL-31 activity byadministrating humanized IL-31 antigen binding molecules or IL-31antagonists is a potential therapeutic approach for IBD.

4. Psoriasis

Psoriasis is a chronic skin condition that affects more than sevenmillion Americans. Psoriasis occurs when new skin cells grow abnormally,resulting in inflamed, swollen, and scaly patches of skin where the oldskin has not shed quickly enough. Plaque psoriasis, the most commonform, is characterized by inflamed patches of skin (“lesions”) toppedwith silvery white scales. Psoriasis may be limited to a few plaques orinvolve moderate to extensive areas of skin, appearing most commonly onthe scalp, knees, elbows and trunk Although it is highly visible,psoriasis is not a contagious disease. The pathogenesis of the diseasesinvolves chronic inflammation of the affected tissues. Humanized IL-31antigen binding molecules or IL-31 antagonists could serve as a valuabletherapeutic to reduce inflammation and pathological effects inpsoriasis, other inflammatory skin diseases, skin and mucosal allergies,and related diseases.

Psoriasis is a T-cell mediated inflammatory disorder of the skin thatcan cause considerable discomfort. It is a disease for which there is nocure and affects people of all ages. Psoriasis affects approximately twopercent of the populations of European and North America. Althoughindividuals with mild psoriasis can often control their disease withtopical agents, more than one million patients worldwide requireultraviolet or systemic immunosuppressive therapy. Unfortunately, theinconvenience and risks of ultraviolet radiation and the toxicities ofmany therapies limit their long-term use. Moreover, patients usuallyhave recurrence of psoriasis.

IL-31 was isolated from tissue known to have important immunologicalfunction and which contain cells that play a role in the immune system.IL-31 is expressed in CD3+ selected, activated peripheral blood cells,and it has been shown that IL-31 expression increases after T cellactivation. Moreover, humanized IL-31 antigen binding molecules or IL-31antagonists can have an effect on the growth/expansion ofmonocytes/macrophages, T-cells, B-cells, NK cells and/or differentiatedstate of monocytes/macrophages, T-cells, B-cells, NK cells or thesecells' progenitors. Factors that both stimulate proliferation ofhematopoietic progenitors and activate mature cells are generally known,however, proliferation and activation can also require additional growthfactors. For example, it has been shown that IL-7 and Steel Factor(c-kit ligand) were required for colony formation of NK progenitors.IL-15+IL-2 in combination with IL-7 and Steel Factor was more effective(Mrozek et al., Blood 87:2632-2640, 1996). However, unidentifiedcytokines may be necessary for proliferation of specific subsets of NKcells and/or NK progenitors (Robertson et. al., Blood 76:2451-2438,1990). Similarly, IL-31 may act alone or in concert or synergy withother cytokines to enhance growth, proliferation expansion andmodification of differentiation of monocytes/macrophages, T-cells,B-cells or NK cells.

The present invention is directed toward use of humanized IL-31 antigenbinding molecules or IL-31 antagonists as antagonists in inflammatoryand immune diseases or conditions such as atopic dermatitis, pruriticdiseases, pancreatitis, type I diabetes (IDDM), pancreatic cancer,pancreatitis, Graves Disease, inflammatory bowel disease (IBD), Crohn'sDisease, colon and intestinal cancer, diverticulosis, autoimmunedisease, sepsis, organ or bone marrow transplant; inflammation due totrauma, surgery or infection; amyloidosis; splenomegaly; graft versushost disease; and where inhibition of inflammation, immune suppression,reduction of proliferation of hematopoietic, immune, inflammatory orlymphoid cells, macrophages, T-cells (including Th1 and Th2 cells, CD4+and CD8+ cells), suppression of immune response to a pathogen orantigen. Moreover the presence of IL-31RA expression in activated immunecells such as activated CD4+ and CD19+ cells showed that IL-31RAreceptor may be involved in the body's immune defensive reactionsagainst foreign invaders: such as microorganisms and cell debris, andcould play a role in immune responses during inflammation and cancerformation. As such, antibodies and binding partners of the presentinvention that are agonistic or antagonistic to IL-31RA receptorfunction, such as IL-31, can be used to modify immune response andinflammation.

Humanized IL-31 antigen binding molecules or IL-31 antagonists may alsobe used within diagnostic systems for the detection of circulatinglevels of IL-31. Within a related embodiment, antibodies or other agentsthat specifically bind to IL-31 polypeptides can be used to detectcirculating IL-31 polypeptides. Elevated or depressed levels of ligandpolypeptides may be indicative of pathological conditions, includingcancer. IL-31 polypeptides may contribute to pathologic processes andcan be an indirect marker of an underlying disease.

In atherosclerotic lesions, one of the first abnormalities islocalization of monocyte/macrophages to endothelial cells. These lesionscould be prevented by use of antagonists to IL-31. Humanized IL-31antigen binding molecules or IL-31 antagonists can be used asantagonists of IL-31 in atherosclerotic lesions. Moreover, monoblasticleukemia is associated with a variety of clinical abnormalities thatreflect the release of the biologic products of the macrophage, examplesinclude high levels of lysozyme in the serum and urine and high fevers.Moreover, such leukemias exhibit an abnormal increase of monocyticcells. These effects could possibly be prevented by antagonists toIL-31, such as those described herein. Moreover, humanized IL-31 antigenbinding molecules or IL-31 antagonists can be conjugated to moleculessuch as toxic moieties and cytokines, as described herein to direct thekilling of leukemia monocytic cells.

IL-31 has been shown to be expressed in activated mononuclear cells, andmay be involved in regulating inflammation. As such, polypeptides of thepresent invention can be assayed and used for their ability to modifyinflammation, or can be used as a marker for inflammation. Methods todetermine proinflammatory and antiinflammatory qualities of IL-31 areknown in the art and discussed herein. Moreover, it may be involved inup-regulating the production of acute phase reactants, such as serumamyloid A (SAA), α1-antichymotrypsin, and haptoglobin, and thatexpression of IL-31RA receptor ligand may be increased upon injection oflipopolysaccharide (LPS) in vivo that are involved in inflammatoryresponse (Dumoutier, L. et al., Proc. Nat'l. Acad. Sci. 97:10144-10149,2000). Production of acute phase proteins, such as SAA, is considered sshort-term survival mechanism where inflammation is beneficial; however,maintenance of acute phase proteins for longer periods contributes tochronic inflammation and can be harmful to human health. For review, seeUhlar, C M and Whitehead, A S, Eur. J. Biochem. 265:501-523, 1999, andBaumann H. and Gauldie, J. Immunology Today 15:74-80, 1994. Moreover,the acute phase protein SAA is implicated in the pathogenesis of severalchronic inflammatory diseases, is implicated in atherosclerosis andrheumatoid arthritis, and is the precursor to the amyloid A proteindeposited in amyloidosis (Uhlar, C M and Whitehead, supra.). Thus, wherea ligand such as IL-31 that acts as a pro-inflammatory molecule andinduces production of SAA, humanized IL-31 antigen binding molecules orIL-31 antagonists would be useful in treating inflammatory disease andother diseases associated with acute phase response proteins induced bythe ligand. For example, a method of reducing inflammation comprisesadministering to a mammal with inflammation or itch an amount of acomposition of humanized IL-31 antigen binding molecules or IL-31antagonists that is sufficient to reduce the inflammation or itch.Moreover, a method of suppressing an inflammatory response in a mammalwith inflammation can comprise: (1) determining a level of serum amyloidA protein; (2) administering a composition comprising a humanized IL-31antigen binding molecules or IL-31 antagonists as described herein in anacceptable pharmaceutical carrier; (3) determining a post administrationlevel of serum amyloid A protein; (4) comparing the level of serumamyloid A protein in step (1) to the level of serum amyloid A protein instep (3), wherein a lack of increase or a decrease in serum amyloid Aprotein level is indicative of suppressing an inflammatory response.

Tissue distribution of the mRNA corresponding it's IL-31RA receptor cDNAshowed that mRNA level was highest in monocytes and prostate cells, andis elevated in activated monocytes, and activated CD4+, activated CD8+,and activated CD3+ cells. Hence, IL-31RA receptor is also implicated ininducing inflammatory and immune response. Thus, particular embodimentsof the present invention are directed toward use of humanized IL-31antigen binding molecules or IL-31 antagonists in inflammatory andimmune diseases or conditions such as, pancreatitis, type I diabetes(IDDM), pancreatic cancer, pancreatitis, Graves Disease, inflammatorybowel disease (IBD), Crohn's Disease, colon and intestinal cancer,diverticulosis, autoimmune disease, sepsis, organ or bone marrowtransplant; inflammation due to trauma, surgery or infection;amyloidosis; splenomegaly; graft versus host disease; and whereinhibition of inflammation, immune suppression, reduction ofproliferation of hematopoietic, immune, inflammatory or lymphoid cells,macrophages, T-cells (including Th1 and Th2 cells, CD4+ and CD8+ cells),suppression of immune response to a pathogen or antigen. Moreover thepresence of IL-31RA receptor and IL-31 expression in activated immunecells such as activated CD3+, monocytes, CD4+ and CD19+ cells showedthat IL-31RA receptor may be involved in the body's immune defensivereactions against foreign invaders: such as microorganisms and celldebris, and could play a role in immune responses during inflammationand cancer formation. As such, humanized IL-31 antigen binding moleculesor IL-31 antagonists of the present invention that are agonistic orantagonistic to IL-31RA receptor function, can be used to modify immuneresponse and inflammation.

Humanized IL-31 antigen binding molecules or IL-31 antagonists areuseful to:

1) Antagonize or block signaling via IL-31RA-comprising receptors in thetreatment of acute inflammation, inflammation as a result of trauma,tissue injury, surgery, sepsis or infection, and chronic inflammatorydiseases such as asthma, inflammatory bowel disease (IBD), chroniccolitis, splenomegaly, rheumatoid arthritis, recurrent acuteinflammatory episodes (e.g., tuberculosis), and treatment ofamyloidosis, and atherosclerosis, Castleman's Disease, asthma, and otherdiseases associated with the induction of acute-phase response; and

2) Antagonize or block signaling via the IL-31RA receptor receptors inthe treatment of autoimmune diseases such as IDDM, multiple sclerosis(MS), systemic Lupus erythematosus (SLE), myasthenia gravis, rheumatoidarthritis, and IBD to prevent or inhibit signaling in immune cells (e.g.lymphocytes, monocytes, leukocytes) via IL-31RA receptor (Hughes C etal, J. Immunol 153: 3319-3325, 1994). Alternatively antibodies, such asmonoclonal antibodies (MAb) to IL-31, can also be used as an antagonistto deplete unwanted immune cells to treat autoimmune disease. Asthma,allergy and other atopic disease may be treated with an MAb against, forexample, anti-IL-31 antibodies, soluble IL-31RA receptor solublereceptors or IL-31RA/CRF2-4 heterodimers, to inhibit the immune responseor to deplete offending cells. Blocking or inhibiting signaling viaIL-31RA, using the polypeptides and antibodies of the present invention,may also benefit diseases of the pancreas, kidney, pituitary andneuronal cells. IDDM, NIDDM, pancreatitis, and pancreatic carcinoma maybenefit. IL-31RA may serve as a target for MAb therapy of cancer wherean antagonizing MAb inhibits cancer growth and targets immune-mediatedkilling (Holliger P, and Hoogenboom, H: Nature Biotech. 16: 1015-1016,1998). Mabs to soluble IL-31RA receptor monomers, homodimers,heterodimers and multimers may also be useful to treat nephropathiessuch as glomerulosclerosis, membranous neuropathy, amyloidosis (whichalso affects the kidney among other tissues), renal arteriosclerosis,glomerulonephritis of various origins, fibroproliferative diseases ofthe kidney, as well as kidney dysfunction associated with SLE, IDDM,type II diabetes (NIDDM), renal tumors and other diseases.

Generally, the dosage of administered humanized IL-31 antigen bindingmolecules or IL-31 antagonists will vary depending upon such factors asthe patient's age, weight, height, sex, general medical condition andprevious medical history. Typically, it is desirable to provide therecipient with a dosage of IL-31 polypeptide which is in the range offrom about 1 pg/kg to 10 mg/kg (amount of agent/body weight of patient),although a lower or higher dosage also may be administered ascircumstances dictate. One skilled in the art can readily determine suchdosages, and adjustments thereto, using methods known in the art.

Administration of a humanized IL-31 antigen binding molecules or IL-31antagonists to a subject can be topical, inhaled, intravenous,intraarterial, intraperitoneal, intramuscular, subcutaneous,intrapleural, intrathecal, by perfusion through a regional catheter, orby direct intralesional injection. When administering therapeuticproteins by injection, the administration may be by continuous infusionor by single or multiple boluses.

Additional routes of administration include oral, mucosal-membrane,pulmonary, and transcutaneous. Oral delivery is suitable for polyestermicrospheres, zein microspheres, proteinoid microspheres,polycyanoacrylate microspheres, and lipid-based systems (see, forexample, DiBase and Morrel, “Oral Delivery of MicroencapsulatedProteins,” in Protein Delivery: Physical Systems, Sanders and Hendren(eds.), pages 255-288 (Plenum Press 1997)). The feasibility of anintranasal delivery is exemplified by such a mode of insulinadministration (see, for example, Hinchcliffe and Illum, Adv. DrugDeliv. Rev. 35:199 (1999)). Dry or liquid particles comprising IL-31 canbe prepared and inhaled with the aid of dry-powder dispersers, liquidaerosol generators, or nebulizers (e.g., Pettit and Gombotz, TIBTECH16:343 (1998); Patton et al., Adv. Drug Deliv. Rev. 35:235 (1999)). Thisapproach is illustrated by the AERX diabetes management system, which isa hand-held electronic inhaler that delivers aerosolized insulin intothe lungs. Studies have shown that proteins as large as 48,000 kDa havebeen delivered across skin at therapeutic concentrations with the aid oflow-frequency ultrasound, which illustrates the feasibility oftrascutaneous administration (Mitragotri et al., Science 269:850(1995)). Transdermal delivery using electroporation provides anothermeans to administer a molecule having IL-31 binding activity (Potts etal., Pharm. Biotechnol. 10:213 (1997)).

A pharmaceutical composition comprising humanized IL-31 antigen bindingmolecules or IL-31 antagonists having IL-31 binding activity can beformulated according to known methods to prepare pharmaceutically usefulcompositions, whereby the therapeutic proteins are combined in a mixturewith a pharmaceutically acceptable carrier. A composition is said to bea “pharmaceutically acceptable carrier” if its administration can betolerated by a recipient patient. Sterile phosphate-buffered saline isone example of a pharmaceutically acceptable carrier. Other suitablecarriers are well-known to those in the art. See, for example, Gennaro(ed.), Remington's Pharmaceutical Sciences, 19th Edition (MackPublishing Company 1995).

For purposes of therapy, molecules having IL-31 binding activity and apharmaceutically acceptable carrier are administered to a patient in atherapeutically effective amount. A combination of a protein,polypeptide, or peptide having IL-31 binding activity and apharmaceutically acceptable carrier is said to be administered in a“therapeutically effective amount” if the amount administered isphysiologically significant. An agent is physiologically significant ifits presence results in a detectable change in the physiology of arecipient patient. For example, an agent used to treat inflammation isphysiologically significant if its presence alleviates at least aportion of the inflammatory response.

A pharmaceutical composition comprising humanized IL-31 antigen bindingmolecules or IL-31 antagonists can be furnished in liquid form, in anaerosol, or in solid form. Liquid forms, are illustrated by injectablesolutions, aerosols, droplets, topological solutions and oralsuspensions. Exemplary solid forms include capsules, tablets, andcontrolled-release forms. The latter form is illustrated by miniosmoticpumps and implants (Bremer et al., Pharm. Biotechnol. 10:239 (1997);Ranade, “Implants in Drug Delivery,” in Drug Delivery Systems, Ranadeand Hollinger (eds.), pages 95-123 (CRC Press 1995); Bremer et al.,“Protein Delivery with Infusion Pumps,” in Protein Delivery: PhysicalSystems, Sanders and Hendren (eds.), pages 239-254 (Plenum Press 1997);Yewey et al., “Delivery of Proteins from a Controlled Release InjectableImplant,” in Protein Delivery: Physical Systems, Sanders and Hendren(eds.), pages 93-117 (Plenum Press 1997)). Other solid forms includecreams, pastes, other topological applications, and the like.

The humanized IL-31 antigen binding molecules or IL-31 antagonistsdisclosed herein may also be formulated as immunoliposomes. Liposomescontaining the antibody are prepared by methods known in the art, suchas described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688(1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030 (1980); andU.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhancedcirculation time are disclosed in U.S. Pat. No. 5,013,556.

Liposomes provide one means to deliver therapeutic polypeptides to asubject intravenously, intraperitoneally, intrathecally,intramuscularly, subcutaneously, or via oral administration, inhalation,or intranasal administration. Liposomes are microscopic vesicles thatconsist of one or more lipid bilayers surrounding aqueous compartments(see, generally, Bakker-Woudenberg et al., Eur. J. Clin. Microbiol.Infect. Dis. 12 (Suppl. 1): 561 (1993), Kim, Drugs 46:618 (1993), andRanade, “Site-Specific Drug Delivery Using Liposomes as Carriers,” inDrug Delivery Systems, Ranade and Hollinger (eds.), pages 3-24 (CRCPress 1995)). Liposomes are similar in composition to cellular membranesand as a result, liposomes can be administered safely and arebiodegradable. Depending on the method of preparation, liposomes may beunilamellar or multilamellar, and liposomes can vary in size withdiameters ranging from 0.02 μm to greater than 10 μm. A variety ofagents can be encapsulated in liposomes: hydrophobic agents partition inthe bilayers and hydrophilic agents partition within the inner aqueousspace(s) (see, for example, Machy et al., Liposomes In Cell Biology AndPharmacology (John Libbey 1987), and Ostro et al., American J. Hosp.Pharm. 46:1576 (1989)). Moreover, it is possible to control thetherapeutic availability of the encapsulated agent by varying liposomesize, the number of bilayers, lipid composition, as well as the chargeand surface characteristics of the liposomes.

Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded-throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al. J. Biol. Chem.257: 286-288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent (such as Doxorubicin) is optionally containedwithin the liposome. See Gabizon et al. J. National Cancer Inst. 81(19)1484 (1989).

Therapeutic formulations of the humanized IL-31 antigen bindingmolecules or IL-31 antagonists are prepared for storage by mixing theantibody having the desired degree of purity with optionalphysiologically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as Tween™, Pluronics™ or polyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, it may be desirable to further provide antibodies whichbind to IL-31 in the one formulation. Alternatively, or in addition, thecomposition may comprise a chemotherapeutic agent or a cytokine. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended.

Polypeptides having IL-31 binding activity can be encapsulated withinliposomes using standard techniques of protein microencapsulation (see,for example, Anderson et al., Infect. Immun 31:1099 (1981), Anderson etal., Cancer Res. 50:1853 (1990), and Cohen et al. Biochim Biophys. Acta1063:95 (1991), Alving et al. “Preparation and Use of Liposomes inImmunological Studies,” in Liposome Technology, 2nd Edition, Vol. III,Gregoriadis (ed.), page 317 (CRC Press 1993), Wassef et al., Meth.Enzymol. 149:124 (1987)). As noted above, therapeutically usefulliposomes may contain a variety of components. For example, liposomesmay comprise lipid derivatives of poly(ethylene glycol) (Allen et al.,Biochim Biophys. Acta 1150:9 (1993)).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly (2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and .gamma.ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the Lupron Depot™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(+3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

Other dosage forms can be devised by those skilled in the art, as shown,for example, by Ansel and Popovich, Pharmaceutical Dosage Forms and DrugDelivery Systems, 5^(th) Edition (Lea & Febiger 1990), Gennaro (ed.),Remington's Pharmaceutical Sciences, 19^(th) Edition (Mack PublishingCompany 1995), and by Ranade and Hollinger, Drug Delivery Systems (CRCPress 1996).

As an illustration, pharmaceutical compositions may be supplied as a kitcomprising a container that comprises a humanized IL-31 antigen bindingmolecule or IL-31 antagonist (e.g., an antibody or antibody fragmentthat binds a IL-31 polypeptide). Therapeutic polypeptides can beprovided in the form of an injectable solution for single or multipledoses, or as a sterile powder that will be reconstituted beforeinjection. Alternatively, such a kit can include a dry-powder disperser,liquid aerosol generator, or nebulizer for administration of atherapeutic polypeptide.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1 Determination of Humanized Variable Region Sequences

Mouse anti-human IL-31 monoclonal antibodies are described in co-pendingU.S. patent application Ser. No. 11/430,066, filed May 8, 2006 (U.S.Patent Publication No. 2006-02752960). The amino acid sequences of thevariable regions of mouse anti-human IL-31 antibodies described inco-pending U.S. patent application Ser. No. 11/850,006, filed Sep. 4,2007 and co-owned PCT application US07/77555, filed Sep. 4, 2007. Theseamino acid sequences were used as starting material for the humanizedsequences described herein. In specific the mouse anti-human IL-31antibody sequence was from a hybridoma with the clone number 292.12.3.1.

Nucleotides encoding the CDR regions of clones 292.12.3.1 were clonedinto a cDNA vector encoding human IgG such that a chimeric antibodieswere generated consisting of a human IgG framework harboring the murineCDR regions. Individual amino acids in the chimeric antibody wereoptimized to obtain the characteristics of a monoclonal antibody of highquality (binding affinity, stability, and homogenecity). Threedimensional models of each antibody were generated and humanizedvariable regions and CDR regions were determined.

Constructs containing the humanized mouse anti-human IL-31 heavy andlight chain variable regions were fused to a human IgG4 constant regionwith a Ser to Pro mutation at position 241 (Kabat numbering) to inhibitformation of half antibodies. The constructs were expressed in HEK2923cells and purified on a protein A column followed by buffer exchangeinto PBS. Binding affinity was measured by Biacore. Potency was measuredin a BAF proliferation assay and a NHK stat3 phosporylation assay.Biophysical characteristics such as homogenecity, aggregation, andfolding stability were evaluated.

Example 2 Expression of Humanized IL-31 Antigen Binding Molecules inHEK293 Cells

All experimental procedures were carried out according to themanufactures instructions. Anti IL-31 antibody cDNA was ordered fromGeneart (http://www.Geneart.com) and subcloned into the expressionplasmid pTT5. Antibody light chain (LC) and heavy chain (HC) were kepton independent plasmids. pTT5 was licensed from Yves Durocher(Biotechnology Research Institute, National Research Council Canada).The pTT5 plasmid has been characterized in the following publications;Durocher et al. NAR 2002; and Pham et al. Biotechn. Bioeng. 2003.

To obtain plasmid for HEK293-6E transfection, anti IL-31 antibody codingplasmids were chemically transformed into TOP10 E. coli cells (cat no.C4040-06, Invitrogen, Taastrup, Denmark) and isolated via plasmidpurification columns (cat no. 27144, Qiagen, Ballerup, Denmark)Site-directed amino acid exchange was performed by PCR, with primersfrom DNA technology, and Dpnl digest (cat no. 200518, Clontech viaMedinova Scientific A/S, Glostrup, Denmark). Plasmids were sequenced atMWG-biotech.

HEK293-6E cells were transfected as described in the 293Fectin protocol(cat no. 12347019, Invitrogen, Taastrup, Denmark). In brief, 15 μg of LCand 15 μg of HC plasmid were diluted in 1 ml of Opti-MEM (cat no.31985-062, Gibco/Invitrogen, Taastrup, Denmark) and mixed with 40 μl of293Fectin also diluted in 1 ml Opti-MEM. For a standard transfection 30million HEK293-6E cells were pelleted and resuspended in 28 ml ofFreestyle medium (cat no. 12338, Gibco/Invitrogen, Taastrup, Denmark)and the 2 ml plasmid mix was then added. The cells were hereafterincubated at 37° C., 8% CO2, with agitation (125 rpm) for 6 days beforepelleting and supernatant sampling.

Example 3 Binding Affinity of Humanized IL-31 Antigen Binding MoleculesMeasured by Biacore

Introduction

Protein interactions can be monitored in real time using surface plasmonresonance (SPR) analysis. In this study we perform SRP analysis onBiacore 3000 and Biacore T100 instruments, in order to characterize theanti-IL-31 monoclonal antibodies with respect to affinity towardsrecombinant human IL-31 (hIL-31).

Affinity studies were performed using a direct binding procedure, withthe monoclonal antibody covalently coupled via free amine groups to thecarboxymethylated dextrane membrane (CM5) on the sensor chip surface.Recombinant hIL-31 was injected in various concentrations, followed by adissociation period with constant buffer flow over the sensor chipsurface. Using this experimental design, the binding of hIL-31 to theimmobilized monoclonal antibody can be regarded as a 1:1 binding, withone hIL-31 molecule binding to one antibody binding site. The kineticparameters for the interaction can be calculated using a 1:1 interactionlangmuir fitting model.

Method

The purified monoclonal antibodies were immobilized in individual flowcells on a CM5 type sensor chip Immobilizations were performed using astandard amine coupling procedure, aiming for an immobilization level of500 Resonance Units (RU). The antibodies were diluted to 1-5 ug/ml in 10mM NaAc pH 4.5.

HPS-EP pH 7.4 (10 mM HEPES, 150 mM NaCl, 3 mM EDTA and 0.005% PolysorbatP20) was used as running buffer, and diluents for the recombinant hIL-31(hIL-31, BHK produced, A1277F, zcytor17lig CEE). The hIL-31 was testedin a 3-fold dilution series from 333.3 nM to 1.4 nM. Association(injection) was 4 min., followed by a 20 min dissociation (wash) period.Flow rate was 50 ul/min. Experiments were performed at 25° C.Regeneration of the surface was accomplished by injection of 30 sec.pulse of 10 mM Glycin-HCl pH 1.8 or 1M formic acid, at a 30 ul/min flowrate. Detection in all flow cells simultaneously. Flow cell #1 containedno immobilized antibody, and was used for subtraction of background andbulk. All experiments were performed in triplicates.

The “parent” murine monoclonal antibody was included in all experimentsfor internal referencing of the kinetic parameters obtained.

The kinetic parameters were calculated by global fitting of the datausing a 1:1 langmuir binding model. Data was inspected formass-transport limitations prior to calculation of the kineticparameters. In some experiments the Rmax was fitted locally, and the RIconstant at 0.

Experiments were performed on Biacore 3000 and T100 instruments. Datawas evaluated using Biaeval 4.1 and Biacore T100 evaluation software.Data is shown in Table 2 and below. Several of the clones showedaffinity similar to the parent strain in this assay.

TABLE 2 KD Relative potency Relative potency Clone Number (nM) kd (l/s)ka (l/Ms) loss (KD) loss (kd) 292.12.3.1 (parent) 2.2 6.10E−05 2.80E+047 1.9 5.70E−05 3.30E+04 Similar to parent Similar to parent 8 1.95.40E−05 2.80E+04 Similar to parent Similar to parent 9 2.1 9.00E−054.30E+04 Similar to parent ~1.5x to parent 10 186 1.50E−02 8.80E+05 ~85xto parent ~245x to parent 11 1.46 7.60E−05 5.20E+04 Similar to parentSimilar to parent 13 24 3.00E−03 1.20E+05 ~10x to parent ~50x to parent14 12.5 7.30E−04 5.90E+04 ~5.5x to parent ~12x to parent 16 1.2 7.50E−056.10E+04 ~0.5x to parent Similar to parent 17 0.9 6.50E−05 7.00E+04~0.4x to parent Similar to parent 18 1.13 6.20E−05 5.50E+04 ~0.5x toparent Similar to parent 21 1.4 9.70E−05 6.90E+04 Similar to parentSimilar to parent 22 1.7 1.10E−04 6.70E+04 Similar to parent ~2x toparent 23 1.4 9.00E−05 6.50E+04 ~0.6x to parent ~1.5x to parent 25 0.857.20E−05 8.50E+04 ~0.5x to parent Similar to parent 26 0.68 7.00E−051.00E+05 ~0.4x to parent Similar to parent 27 0.8 7.90E−05 9.80E+04~0.5x to parent ~1.3x to parent 28 0.46 5.60E−05 1.20E+05 ~0.25x toparent Similar to parent 29 0.99 8.98E−05 9.00E+04 ~0.5x to parent ~1.5xto parent 30 1.1 9.00E−05 7.80E+04 ~0.5x to parent ~1.5x to parent 310.77 7.00E−05 9.20E+04 ~0.4x to parent Similar to parent 32 0.645.90E−05 9.30E+04 ~0.3x to parent Similar to parent 33 1.3 6.80E−055.20E+04 ~0.6x to parent Similar to parent 34 1.4 6.90E−05 5.20E+04~0.6x to parent Similar to parent 35 1.6 1.00E−04 6.40E+04 ~0.7x toparent ~1.5x to parent 36 1.6 1.08E−04 6.80E+04 ~0.5x to parent ~1.5x toparent

Example 4 Potency of Humanized IL-31 Antigen Binding Molecules Measuredby BAF Proliferation Assay

A. Media and Buffers

Culture medium: RPMI 1640 with Glutamax (SKN, NN), 10% heat inactivatedFBS, 1% P/S (BioWhitaker Cat. No. DE17-602E), 0.5 mg/ml Geneticin (GIBCOCat. No. 10131-019), 100 μg/ml Zeocin (Invitrogen 45-0430), 1 ng/mlmouse IL3 (TriChem ApS Cat. No. 213-13), 2 ug/ml Pyromycin(Sigma-Aldrich P7255).

Assay medium: RPMI 1640 with Glutamax (SKN, NN), 10% heat inactivatedFBS, 1% P/S (BioWhitaker Cat. No. DE17-602E), 0.5 mg/ml Geneticin (GIBCOCat. No. 10131-019), 100 μg/ml Zeocin (Invitrogen 45-0430), 2 ug/mlPyromycin (Sigma-Aldrich P7255).

alamarBlue dye (BioScource, Da11100) is used to assess proliferation.

B. Antibodies, Cells and Cytokines

Human anti-IL-31 monoclonal antibodies were produced at Novo Nordisk andpurified at Novo Nordisk (Copenhagen, Denmark). BAF-3(hIL-31R) cellsreceived from ZymoGenetics, Inc. (Seattle, Wash.) as a KZS134-BAF3 cellline transfected with the genes for hIL-31Rα and hOSMRB. Recombinanthuman IL-31 (C108S, described in U.S Patent Publication No.2006-0228329), produced in E. Coli by ZymoGenetics, Inc.). MW 18 kDa

C. Proliferation Assay

1. Stimulation Assay

BAF-3(hIL-31R) cells are washed thoroughly in Assay medium to get rid ofresidual IL3. The cells are then seeded into 96-well microtiter plates(flat-well view plate Packard cat.S00190) at 10⁴ cells per well. Serialdilutions of hIL-31 (10⁻⁹ M to 10⁻¹⁵ M) are added to the wells andadditional wells with cells but no hIL-31 serves as negative control.The cells are cultured for three days in 5% CO2 at 37° C. For the last 6hours of the culture period, 101.1.1 alamarBlue is added to each well.The cells are analyzed for fluorescence intensity on aspectrofluorometer (bmg POLARstar+ Galaxy) at excitation 555-12 nm andemission 590 nm. For inhibition analysis, a constant concentration ofhIL-31 is used to stimulate the cells. This concentration was chosen onbasis of approximately 90% of max stimulation in the proliferation assaywhich in our hands means 10⁴⁰ M hIL20.

2. Inhibition Assay.

10×10⁴ cells per well of washed BAF-3(hIL-31R) cells are seeded intomicrotiter wells in assay medium. 10-10M (final concentration) of hIL-31is added to each well (except some wells used as negative controlcontaining only cells. Serial dilutions of antibody (i.e. 100 μg and2-fold) are added to the wells already containing cells and cytokines(except wells used for positive controls which should contain onlycells+hIL-31). The mixture of cells, cytokine and antibody are incubatedin 1000w for 72 hours in 5% CO2 at 37° C. The last 6 hours of incubationincludes 100w of alamarBlue. The plates are analysed for fluorescenceintensity on a spectrofluorometer (bmg POLARstar+ Galaxy) at excitation555-12 nm and emission 590 nm. The curves are drawn and the potency(IC50) is calculated using Prism 4 (GraphPad PRISM software Inc.). Datais shown in Table 3 and below. Several of the clones showed potencysimilar to the parent in this assay.

TABLE 3 Potency (nM) of humanised mAbs at 1E−10M hIL-31 Average Foldloss in potency Clone Number Average Potency relative to 292.12.3.1292.12.3.1 (parent) 1.6 1.0 07 4.5 3.3 08 4.1 2.5 09 28.5 15.1 10 >> >>11 22.8 11.6 12 >> >> 13 109.0 99.1 14 >> >> 15 10.4 7.7 16 10.6 9.8 175.9 5.2 18 8.1 5.9 20 >> >> 21 4.8 3.5 22 14.0 13.2 23 8.6 7.6 24 6.04.0 25 26.3 16.5 26 9.3 5.8 27 29.1 18.7 28 26.4 17.1 29 12.9 8.3 3057.8 37.1 31 32.4 25.1 32 18.9 14.7 33 3.1 2.3 35 62.1 75.1 36 14.1 —

Example 5 Potency of Humanized IL-31 Antigen Binding Molecules Measuredby NHK Stat3 Phosphorylation Assay

Culture of Normal Human Keratinocytes and STAT3 Phosphorylation Assay:

Normal human keratinocytes (NHKs) from abdominal skin were obtained fromBiopredic Int. (Rennes, France) and were grown in Epilife Mediumsupplemented with the HKGS kit from Cascade Biologics (Portland, Oreg.).A Detach Kit containing HBSS, trypsin-EDTA and trypsin neutralizingreagent was used to harvest cells and was obtained from Promocell(Heidelberg, Germany) NHKs were stimulated for 15 minutes withrecombinant human IL-31 in flat-bottomed 96-well plates (Nunc, Roskilde,Denmark) in order to measure STAT3 phosphorylation and determine theEC50 of the cytokine. NHK lysates were tested in PathScan Phospho-STAT3Sandwich ELISA Kit from Cell Signaling Technology (Danvers, Mass.)according to the manufacturer's instructions. The IC50 of eachneutralizing antibody was then determined by adding serial dilutions ofantibodies to NHKs prior to stimulation for 15 minutes with an EC80 ofrecombinant human IL-31. Mouse IgG1 (R&D Systems, Minneapolis, Minn.)and human IgG4 (Sigma-Aldrich, Saint Louis, Mo.) were used as isotypiccontrol antibodies.

Potencies of Antibodies Directed Against Human IL-31

For each antibody directed against human IL-31, IC50 values (nM) areshown in Table 4. For each experiment, a Z′ factor as determined withthe XL Fit software is given. The stimulation index obtained with anEC80 of recombinant human IL-31 is shown. Several of the clones showedpotency similar to the parent or acceptable potency in this assay.

TABLE 4 Exp 1 Exp 2 Exp 3 Exp 4 Exp 6 Exp 7 Exp 8 Exp 9 Exp 10Stimulation Index 4.97 4.22 7.61 3.63 6.00 9.05 4.08 6.79 8.70 Z′ factor0.98 0.98 0.93 0.83 0.91 0.93 0.92 0.95 0.90 Clone Number 0.71 1.90 3.668.17 4.48 5.54 5.57 7.06 13.16 292.12.3.1 (parent) 7 7.80 3.99 7.74 5.447.58 8.82 8 6.74 3.44 9 3.25 7.37 6.59 7.10 10 68.83 223.87 13 46.51210.90 14 71.92 242.30 16 3.84 6.22 17 6.65 5.29 18 2.60 2.68 25 5.857.28 26 7.84 6.93 27 3.42 9.59 28 10.50 5.95 29 4.49 5.37 30 14.79 8.7131 11.01 7.82 32 11.53 6.43 33 4.08 5.68 13.62 34 5.46 8.40 35 12.04 368.95

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

What is claimed is:
 1. A pharmaceutical composition comprising amonoclonal antibody that specifically binds to human IL-31 and apharmaceutically acceptable carrier, wherein the monoclonal antibodycomprises a heavy chain variable domain comprising the amino acidsequence of SEQ ID NO:44, and a light chain variable domain comprisingthe amino acid sequence of SEQ ID NO:26, and wherein the composition issuitable for intravenous, intraarterial, intraperitoneal, intramuscularor subcutaneous injection.
 2. The pharmaceutical composition of claim 1,wherein the monoclonal antibody is in a lyophilized form andreconstituted in the pharmaceutically acceptable carrier prior toinjection.
 3. The method according to claim 1, wherein the monoclonalantibody comprises a human Fc domain.
 4. The method according to claim3, wherein the isotype of the human Fc domain is IgG1, IgG2, IgG3 orIgG4.
 5. The method according to claim 3, wherein the isotype of thehuman Fc domain is IgG4.
 6. The method according to claim 5, wherein theIgG4 human Fc domain has a serine to proline mutation at position 241 asdetermined by Kabat.
 7. The method according to claim 1, wherein theheavy chain domain of the monoclonal antibody comprises the amino acidsequence of SEQ ID NO:47.
 8. The method according to claim 1, whereinthe light chain domain of the monoclonal antibody comprises the aminoacid sequence of SEQ ID NO:46.